G-CSF immunoglobulin fusion proteins

ABSTRACT

Disclosed are fusion proteins comprising a biologically active molecule and an immunoglobulin (Ig) Fc domain which is linked to the biologically active molecule. The Fc domain is a hybrid human Fc domain of (i) IgG1, IgG2 or IgG4 or (ii) IgG4 and IgD. The hybrid Fc is useful as a carrier of biologically active molecules.

This is a continuation of U.S. patent application Ser. No. 12/950,500filed Nov. 19, 2010, which is a continuation of U.S. patent applicationSer. No. 12/130,002 filed May 30, 2008 (now issued as U.S. Pat. No.7,867,491), which claims benefit from U.S. provisional application60/940,753 filed on May 30, 2007, the entireties of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a hybrid human Fc and an immunoglobulinfusion protein in which the hybrid human Fc is joined to a biologicallyactive molecule. In particular, it relates to a hybrid human Fc, whichis derived from combinations of human immunoglobulin G (IgG) subclassesor combinations of human IgD and IgG, and a fusion protein in which suchan Fc is coupled to a biologically active molecule via a covalent bond.

RELEVANT ART

Biologically active molecules may be of great interest therapeutically.However, they may have disadvantages as a therapeutic agent becausetheir in vivo stability is low. Their circulating half-life or serumhalf-life is short because they are digested by various enzymes inliving body. Therefore, it has been desired to improve circulatinghalf-life of biologically active molecules.

It has been known that increasing the size of a protein can increase itshalf-life by preventing removal of the protein by kidney (Knauf et al.,J. Biol. Chem. 1988. 263:15064-15070). For example, it was reported toincrease protein stability by coupling an active protein to humanalbumin (Kinstler et al., Pharm. Res. 1995. 12: 1883-1888). However,since the coupling of an active protein to human albumin only slightlyincreases its residence time, it was not an effective method to developan effective pharmaceutical formulation containing the active proteinwhich is coupled to human albumin.

The other reported method is to modulate glycosylation of a protein. Theadditional glycosyltion at the protein and the introduction of sialicacids to the proteins lead to the prevention of degradation of theproteins in liver. But, the increase in glycosylation of the proteinsalso leads to a decrease of bioactivity of the proteins.

To stabilize proteins and prevent clearance by kidney, proteins havebeen conjugated to polyethylene glycol (PEG). The covalent conjugationto PEG has been widely used to deliver a drug of a prolonged half-life(Delgado et al., 1992. 9: 249-304). However, it was reported that PEGconjugation to cytokines or hormones results in a reduced receptorbinding affinity due to steric hindrance caused by the conjugation.

Recently, fusion proteins manufactured using an immunoglobulin (Ig) hasbeen researched and developed. Ig is a major component of blood. HumanIg (hIg) includes various classes such as IgG, IgM, IgA, IgD, and IgE(Roitt et al., “Immunology” 1989, Gower Medical Publishing, London, U.K.; New York, N.Y.). Human IgGs can be further classified into varioussubtypes known as human IgG1 (hIgG1), human IgG2 (hIgG2), human IgG3(hIgG3), and human IgG4 (hIgG4).

Immunoglobulins are comprised of four polypeptide chains, two heavychains and two light chains, which are associated via disulfide bonds toform tetramers. Each chain is composed of a variable region and aconstant region. The constant region of the heavy chain is furtherdivided into three or four regions (CH1, CH2, CH3, and CH4), dependingon the isotypes. The Fc portion of the heavy chain constant region,depending on the Ig isotype, includes hinge, CH2, CH3, and/or CH4domains.

Regarding serum half-life, IgG1, IgG2, and IgG4 have long half-lives of21 days, while other immunoglobulins have relatively short half liveswith less than a week. The chimeric proteins fused to Fc portion of IgGshows increased stability and increased serum half-life (Capon et al.,Nature 1989. 337: 525-531). The biologically active proteins have beenfused at the N-terminus of the CH1 region, the N-terminus of Fc region,or at the C-terminus of CH3 region of IgGs.

At the beginning period, IgG fusion proteins have been created with theextracellular domains of cell surface receptors such as CD4 (Capon etal., Nature 1989. 337: 525-531), TNFR (Mohler et al., J. Immunology1993. 151: 1548-1561), CTLA4 (Linsley et al., J. Exp. Med. 1991. 173:721-730), CD86 (Morton et al., J. Immunology 1996. 156: 1047-1054).Also, there are several cytokines and growth hormones which have beenfused to IgG domains. However, unlike the fusion with the extracellulardomains of cell surface receptors, the fusion with soluble proteins toIgGs leads to reduced biological activities, compared to the non-fusedcytokine or growth factors. The chimeric proteins exist as dimers, whichlead to the steric hindrance from the interacting with their targetmolecules like receptors, due to the presence of two active proteins inclose proximity to one another. Therefore, this problem should beovercome to make an efficient fusion protein.

The other limitation of the Fc fusion technology is the presence ofundesirable immune responses. The Fc domain of the immunoglobulin hasalso effector functions such as antibody dependent cell-mediatedcytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thiseffector functions are generally achieved via interaction between the Fcregion of the Ig and FcRs on effector cells or via complement binding.Therefore, the blocking of effector functions of Fc should be performedto reduce the undesirable reactions such as cell killing, cytokinerelease, or inflammation.

In summary, there are needs for improved Fc fusion proteins with minimalloss of biological activity and with less risk of undesired immuneresponses.

SUMMARY OF THE INVENTION

The present invention provides a hybrid Fc, which is derived fromcombinations of human IgG subclasses or combinations of human IgD andIgG. The hybrid Fc is effective, when joined to a biologically activemolecule, to increase serum half-life of the biologically activemolecule as well as increase expression level of the polypeptide when anucleotide coding for the Fc-polypeptide fusion protein is expressed.

The present invention also provides a hybrid Fc fusion polypeptide inwhich the hybrid Fc is joined to a biologically active molecule. Thefusion protein sometimes is referred to as ‘biologicallyactive-molecule-Fc fusion protein” or simply “fusion protein.” Thefusion protein may have a linker between the Fc and the biologicallyactive molecule. The Fc may be coupled at its N-terminal to a C-terminalof the biologically active molecule.

The fusion protein may be produced by fabricating a nucleotide constructcoding for and being capable of expressing the fusion protein;expressing it in a host cell; and harvest the fusion protein.Alternatively, the fusion protein may be produced by expressing anucleotide coding for the Fc and coupling it to a biologically activemolecule in a conventional manner.

The polypeptide according to one embodiment of the present invention maybe represented by the following formula:N′—(Z1)_(p)-Y—Z2-Z3-Z4-C′

wherein N′ is the N-terminus and C′ is the C-terminus of thepolypeptide;

Z1 indicates an amino acid sequence including at least a C-terminalportion of the amino acid residues at positions 90 to 98 of SEQ ID NO:11or at least a portion of the amino acid residues at positions 90-98 ofSEQ ID NO:14;

Y indicates an amino acid sequence including at least a C-terminalportion of the amino acid residues at positions 99 to 113 of SEQ IDNO:11 or at least a portion of the amino acid residues at positions 99to 162 of SEQ ID NO:14;

Z2 indicates an amino acid sequence including at least an N-terminalportion of the amino acid residues at positions 111 to 147 of SEQ IDNO:12 or at least a portion of the amino acid residues at positions 163to 199 of SEQ ID NO:14;

Z3 indicates an amino acid sequence including at least a C-terminalportion of the amino acid residues at positions 118-223 of SEQ ID NO:11,114-219 of SEQ ID NO:12, 165-270 of SEQ ID NO: 24, or 115 to 220 of SEQID NO:13;

Z4 indicates an amino acid sequence including at least an N-terminalportion of the amino acid residues at positions 224-330 of SEQ ID NO:11,220-326 of SEQ ID NO:12, 271-377 of SEQ ID NO: 24, or 221-327 of SEQ IDNO:13; and

p is an integer of 0 or 1,

wherein the total number of the amino acid residues for Z2 and Z3 isbetween 80 and 140, both inclusive.

Z1 can be an amino acid sequence including 5 to 9 consecutive amino acidresidues from the C-terminal side of the amino acid residues atpositions 90-98 of SEQ ID NO: 11, or 5-9 consecutive amino acid residuesfrom the C-terminal side of the amino acid residues at positions 90-98of SEQ ID NO: 14. In some embodiments, Z1 can be 5, 6, 7, 8 or 9C-terminal amino acid residues of an IgG1 CH1 domain (SEQ ID NO: 11) orIgD CH1 domain (SEQ ID NO: 14).

In another embodiment, Z1 is an amino acid sequence including amino acidresidues at positions 90 to 98 of SEQ ID NO: 11 or amino acid residuesat positions 90 to 98 of SEQ ID NO: 14. Z1 may be an amino acid sequenceconsisting of 5 to 9 amino acid residues at positions 90 to 98 of SEQ IDNO: 11 or amino acid residues at positions 90 to 98 of SEQ ID NO: 14. Z1also may be an amino acid sequence consisting of amino acid residues 90to 98 of SEQ ID NO: 11 or amino acid residues 90 to 98 of SEQ ID NO: 14.

Y can be an amino acid sequence including 5 or more, or 10 or moreconsecutive amino acid residues from the C-terminal side of the aminoacid residues at positions 99 to 113 of SEQ ID NO: 11 or 5 or more, or10 or more consecutive amino acid residues from the C-terminal side ofthe amino acid residues at positions 99 to 162 of SEQ ID NO: 14. Incertain embodiments, Y can be an amino acid sequence including aminoacid residues at positions 99 to 113 of SEQ ID NO: 11, amino acidresidues at positions 158 to 162 of SEQ ID NO: 14, amino acid residuesat positions 153 to 162 of SEQ ID NO: 14, amino acid residues atpositions 143 to 162 of SEQ ID NO: 14, amino acid residues at positions133 to 162 of SEQ ID NO: 14, or amino acid residues at positions 99 to162 of SEQ ID NO: 14.

Z2 can be an amino acid sequence including 4 to 37, or 6-30 consecutiveamino acid residues from the N-terminal side of the amino acid residuesat positions 111 to 147 of SEQ ID NO: 12 (hIgG2) or 4 to 37, or 6-30consecutive amino acid residues from the N-terminal side of the aminoacid residues at positions 163 to 199 of SEQ ID NO: 14 (hIgD). Incertain embodiments, Z2 can be 6 N-terminal amino aid residues of ahuman IgG2 CH2 domain or 8 N-terminal amino acid residues of a human IgDCH2 domain.

The total number of amino acid residues of Z2 and Z3 can be between 80and 140. In an embodiment, the total number of amino acid residues of Z2and Z3 is between 90 and 120, both inclusive. In another embodiment, thetotal number of amino acid residues of Z2 and Z3 is between 105 and 115,both inclusive. In one embodiment, the total number of amino acidresidues of Z2 and Z3 is 108. In a still embodiment, the total number ofamino acid residues of Z2 and Z3 is 109.

Z4 can be an amino acid sequence including 90 or more, or 100 or moreconsecutive amino acid residues at positions 224-330 of SEQ ID NO:11(hIgG1), 220-326 of SEQ ID NO:12 (hIgG2), 271-377 of SEQ ID NO: 24(hIgG3), or 221 to 327 of SEQ ID NO: 13 (hIgG4). Z4 can be an amino acidsequence of the amino acid residues at positions 224-330 of SEQ IDNO:11, 220-326 of SEQ ID NO:12, 271-377 of SEQ ID NO: 24, or 221 to 327of SEQ ID NO: 13.

According to an embodiment, Z3-Z4 is an amino acid sequence selectedfrom the group consisting of (i) a continuous amino acid sequencecomprised of the C-terminal portion of the amino acid residues atpositions 118 to 223 of SEQ ID NO:11 and the N-terminal portion of theamino acid residues at positions 224 to 330 of SEQ ID NO:11, (ii) acontinuous amino acid sequence comprised of the C-terminal portion ofthe amino acid residues at positions 114 to 219 of SEQ ID NO:12 and theN-terminal portion of the amino acid residues at positions 220 to 326 ofSEQ ID NO:12, (iii) a continuous amino acid sequence comprised of theC-terminal portion of the amino acid residues at positions 165 to 270 ofSEQ ID NO: 24 and the N-terminal portion of the amino acid residues atpositions 271 to 377 of SEQ ID NO: 24, and (iv) a continuous amino acidsequence of the C-terminal portion of the amino acid residues atpositions 115 to 220 of SEQ ID NO: 13 and the N-terminal portion of theamino acid residues at positions 221 to 327 of SEQ ID NO:13.

The total number of amino acid residues of the polypeptide according toone embodiment of the present invention is from 154 to 288.

In one embodiment, Y can be an amino acid sequence including at least aportion of the amino acid residues at positions 99-113 of SEQ ID NO: 11,p can be 1 or 0, Z2 can be an amino acid sequence including at least aportion of the amino acid residues at positions 111-147 of SEQ ID NO:12, and Z3 can be an amino acid sequence including at least a portion ofthe amino acid residues at positions 118-223 of SEQ ID NO:11, 114-219 ofSEQ ID NO:12, 165-270 of SEQ ID NO: 24, or 115 to 220 of SEQ ID NO: 13.In this embodiment, when p is 1, Z1 can be an amino acid sequenceincluding at least a portion of the amino acid residues at positions 90to 98 of SEQ ID NO: 11.

In further embodiments, Z3 can be 73 to 106 consecutive amino acidresidues at positions 118-223 of SEQ ID NO:11, 114-219 of SEQ ID NO:12,165-270 of SEQ ID NO: 24, or 115-220 of SEQ ID NO: 13, and the totalnumber of the amino acid residues of Z2 and Z3 can be 110. Z2 can be anamino acid sequence of the amino acid residues at positions 111-116 ofSEQ ID NO: 12, and Z3 can be an amino acid sequence of the amino acidresidues at positions 120-223 of SEQ ID NO:11, 116-219 of SEQ ID NO:12,167-270 of SEQ ID NO: 24, or 118 to 220 of SEQ ID NO: 13.

In another embodiment, Y can be an amino acid sequence including atleast a portion of the amino acid residues at positions 99 to 162 of SEQID NO: 14, p can be 1 or 0 (zero), Z2 can be an amino acid sequenceincluding at least a portion of the amino acid residues at positions 163to 199 of SEQ ID NO: 14, and Z3 can be an amino acid sequence includingat least a portion of the amino acid residues at positions 121 to 220 ofSEQ ID NO: 13. In this embodiment, when p is 1, Z1 can be an amino acidsequence including the amino acid residues at positions 90 to 98 of SEQID NO: 14.

In further embodiments, Y can be 20 consecutive amino acid residues ormore, 30 consecutive amino acid residues or more, 40 consecutive aminoacid residues or more, 50 consecutive amino acid residues or more, or 60consecutive amino acid residues or more of the C-terminal side of theamino acid residues at positions 99-162 of SEQ ID NO: 14. Z2 can be theamino acid residues at positions 163 to 170 of SEQ ID NO: 14, Z3 cancomprise 71 to 100 consecutive amino acid residues of the C-terminalside of the amino acid residues at positions 124-223 of SEQ ID NO:11,120-219 of SEQ ID NO:12, 171-270 of SEQ ID NO: 24, or 121-220 of SEQ IDNO: 13. The total number of the amino acid residues for Z2 and Z3 can be108.

In one embodiment, the polypeptide may be encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:26, and SEQ ID NO:27. The polypeptide is an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, and SEQID NO: 29.

In an embodiment, the polypeptide is fused at its N-terminus with abiologically active molecule which shows an increased circulatinghalf-life compared to the circulating half-life of the native form ofsaid biologically active molecule. The biologically active molecule maybe a polypeptide, protein, or a peptide. The biologically activemolecule may be a polypeptide, peptide or a protein drug. Thebiologically active molecule may be a soluble protein such as, but notlimited to, a hormone, cytokine, growth factor, a co-stimulatorymolecule, hormone receptor, cytokine receptor, growth factor receptor,or short peptide. The biologically active molecule may be EPO or itsvariants/fragments, p40 or its variants/fragments (e.g., p40 variantwhich contains Asn303Gln substitution), G-CSF or its variants/fragments,TNF receptor, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, IL-10 receptor, TGF-beta, TGF-beta receptor, IL-17, IL-17receptor, Factor VII, CXCL-11, FSH, human growth hormone, bonemorphogenetic protein-1 (BMP-1), CTLA4, PD-1, GLP-1, betacellulin, OPG,RNAK, interferon-alpha, interferon-beta or their variants/fragments. Thebiologically active molecule may be a secreted protein, which may be ina mature form.

In one embodiment, there is provided a method of producing thepolypeptide according to claim 1, wherein the method comprises the stepsof: (i) introducing a DNA molecule coding for the polypeptide into amammalian host cell, (ii) growing the cell under conditions where thepolypeptide can be expressed in its growth medium; and (iii) harvestingthe expressed polypeptide. The mammalian host cell may be a CHO, COS orBHK cells.

In another embodiment, there is provided a method of (i) reducing thesymptoms of, preventing or treating an autoimmune disease, (ii)inhibiting rejection of a graft, or (iii) treating or preventingendotoxin-induced shock, including administering a therapeuticallyeffective amount of the polypeptide described above, wherein thepolypeptide is fused to a biologically active molecule.

In one embodiment, there is provided an isolated nucleic acid moleculewhich encodes the polypeptide according to embodiments of the presentinvention. The polypeptide may have an amino acid sequence selected fromthe group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, and SEQ ID NO:29. The nucleic acid molecule may have a nucleotide sequence as shown inSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 26, or SEQ ID NO: 27. The nucleic acid moleculemay further include a signal sequence or leader sequence.

According to an embodiment of the invention, there are provided anexpression vector including the nucleic acid molecule and a host cellcontaining the vector. Examples of the expression vector may include, benot limited to, pAD11 EPO-hFc-1, pAD11 G-CSF-hFc-1, pAD11p40N303Q-hFc-1, pAD11 EPO-hFc-6, pAD11 G-CSF-hFc-6, pAD11p40N303Q-hFc-6, pAD11 EPO-hFc-5, pAD11 G-CSF-hFc-5, pAD11 p40N303Q-hFc-5and pAD11 TNFR-hFc-5.

In one embodiment, there is provided a method of delivering abiologically active molecule to a mammal, including the step ofadministering the nucleic acid molecule to the mammal in need thereof.

In another embodiment, a polypeptide includes an Fc domain whichconsists of a hinge region, a CH2 domain and a CH3 domain in anN-terminal to C-terminal direction, wherein said hinge region includesat least a portion of amino acid residues of a human IgD hinge region orhuman IgG1 hinge region; said CH2 domain includes at least a portion ofamino acid residues of a human IgG4 CH2 domain, wherein 4-37 consecutiveamino acid residues at the N-terminus of the human IgG4 CH2 domain arereplaced with at least a portion of amino acid residues of theN-terminal region of a human IgG2 CH2 domain or the N-terminal region ofa human IgD CH2 domain, and said CH3 domain includes at least a portionof amino acid residues of a human IgG4 CH3 domain.

The hinge region may include at least a portion of amino acid residuesof the human IgG1 hinge region, said CH2 domain includes at least aportion of amino acid residues of the human IgG4 CH2 domain, wherein the4-37 amino acid residues at the N-terminus of the human IgG4 CH2 domainare replaced with at least a portion of the amino acid residues of theN-terminal region of the human IgG2 CH2 domain.

The hinge region may include at least a portion of amino acid residuesof the human IgD hinge region, said CH2 domain includes at least aportion of amino acid residues of the human IgG4 CH2 domain, wherein4-37 amino acid residues at the N-terminus of the human IgG4 CH2 domainare replaced with at least a portion of the amino acid residues of theN-terminal region of the human IgD CH2 domain.

The polypeptide may further include a CH1 domain, wherein said CH1domain includes at least a portion of amino acid residues of the humanIgG1 CH1 domain, and wherein said CH1 domain is coupled to theN-terminus of said hinge region. The polypeptide may further include aCH1 domain, wherein said CH1 domain includes at least a portion of aminoacid residues of the human IgD CH1 domain, and wherein said CH1 domainis coupled to the N-terminus of said hinge region. The polypeptide mayfurther include a second polypeptide coupled to the N-terminus of saidhinge region, wherein the second polypeptide is a biologically activenon-immunoglobulin polypeptide. The polypeptide may further include abiologically active molecule coupled to the N-terminus of said CH1domain or to the C-terminus of said CH4 domain through a linker, whereinsaid biologically active molecule is not an immunoglobulin polypeptide.The polypeptide and the biologically active molecule may be coupled toeach other via a linker. The linker molecule is an albumin linker or asynthetic linker. The albumin linker comprises amino acid sequence 321to 323, 318 to 325, 316 to 328, 313 to 330, 311 to 333, or 306 to 338 ofSEQ ID NO: 25. The synthetic linker may be a peptide of 10 to 20 aminoacid residues composed of Gly and Ser residues. In one embodiment, suchGly-Ser linker is GGGGSGGGGSGGGSG (SEQ ID NO: 32).

The present invention also encompasses an antibody molecule comprising arecombinant Fc region, the recombinant Fc region is described as above.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 shows the schematic diagram of hybrid Fcs (hFcs) that can be usedas a carrier protein for biologically active molecules designated as“X”.

FIG. 2 shows the schematic representations of hFcs following detaileddescription about amino acid positions derived from IgG1 (SEQ ID NO:11), IgG2 (SEQ ID: 12), IgG4 (SEQ ID: 13) and IgD (SEQ ID: 14). The samerule applies to the designation of amino acid positions in thepolypeptide throughout the application, unless otherwise indicated.

FIG. 3 shows the schematic representation of hFcs which each areconjugated to biologically active molecules designated as “X” at theC-terminal through an albumin linker peptide designated as “AL”.

FIG. 4 shows the schematic representations of hFcs conjugated withlinkers following detailed description about amino acid positions ofalbumin linkers derived from human albumin (SEQ ID NO: 25).

FIG. 5 shows the results of hydrophobicity plot of hFc-6.

FIG. 6( a) shows the results of FcγRI binding activities of MabThera®(Rituximab), hIgG1, Enbrel® (etanercept), EPO-hFc-5, G-CSF-hFc-5,p40N303Q-hFc-5 using specific ELISA assay; FIG. 6( b) shows the resultsof C1q binding activities of MabThera® (Rituximab), hIgG1, Enbrel®(etanercept), EPO-hFc-5, G-CSF-hFc-5, p40N303Q-hFc-5 using specificELISA assay.

FIG. 7( a) shows the results of bioactivities of EPO-IgG1 Fc, EPO-hFc-1,EPO-hFc-5, EPO-hFc-6 and Aranesp® (darbepoetin alfa), compared to thatof EPO in human F36E cell line; FIG. 7( b) shows the results of in vitrobioactivities of Neulasta® (pegfilgrastim) and G-CSF-hFc-5 in mousehematopoietic cell line (NFS-60); FIG. 7( c) shows the results of invitro bioactivities of p40 and p40N303Q-hFc-5 in human PBMCs; FIG. 7( d)shows the results of in vitro bioactivities of Enbrel® (etanercept) andTNFR-hFc-5 in Murine L929 cells; and FIG. 7( e) shows the results of invitro bioactivities of thFc-1-AL(0)-IFN-beta and thFc-1-AL(3)-IFN-betain human WISH cells.

FIG. 8( a) shows the results of in vivo half life of Aranesp®(darbepoetin alfa), EPO-hFc-1, or EPO-hFc-5 administered to cynomolgusmonkeys via SC route (left panel) and IV route (right panel); FIG. 8( b)shows the results of pharmacokinetics of LEUCOSTIM® (filgrastim) andG-CSF-hFc-1 administered to Sprague Dawley rats via SC route (leftpanel) and IV route (right panel); FIG. 8( c) shows the results ofpharmacokinetics of p40N303Q-hFc-5 and Enbrel® (etanercept) administeredto cynomolgus monkeys via SC route: FIG. 8( d) shows the results ofpharmacokinetics of TNFR-hFc-5 and Enbrel® (etanercept) administered toSprague Dawley rats via SC route.

FIG. 9( a) shows the results of in vivo bioactivities of Aranesp®(darbepoetin alfa) and EPO-hFc-5 administered to cynomolgus monkeys viaSC route (upper panel) and IV route (lower panel) and FIG. 9( b) showsthe results of in vivo bioactivities of LEUCOSTIM® (filgrastim) andG-CSF-hFc-1 administered to Sprague Dawley rats via SC route (upperpanel) and IV route (lower panel).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a hybrid human immunoglobulin Fcfragment, which includes a hinge region, a CH2 domain and a CH3 domainin an N-terminal to C-terminal direction, wherein the hinge region is anat least partial amino acid sequence of a human IgD hinge region or ahuman IgG1 hinge region; and the CH2 domain is of human IgG4 CH2 domain,a portion of which, at its N-terminal region, is replaced by 4-37 aminoacid residues of an N-terminal region of a human IgG2 CH2 or human IgDCH2 domain. Such hybrid Fc fragment, when joined to a biologicallyactive molecule, such as a biologically active molecule, to produce a Fcfusion protein, minimizes non-specific immunoreactions of the Fc fusionprotein, prolongs the serum half-life of the biologically activemolecule, and optimizes the activity of the biologically activemolecule.

In the Fc fusion protein according to one embodiment of the presentinvention, the combination of the N-terminal of IgD CH2 domain with theremaining portion of the IgG4 CH2 domain was designed that the region ofthe resulting fusion protein where two different Ig subunits arerecombined is hydrophobic. The hydrophobic region of the resulting fusedprotein will be located inside a folded protein, minimizing undesirednon-specific immune reaction.

The term “Fc fragment” or “Fc,” as used herein, refers to a protein thatcontains the heavy-chain constant region 1 (CH1), the heavy-chainconstant region 2 (CH2) and the heavy-chain constant region 3 (CH3) ofan immunoglobulin, and not the variable regions of the heavy and lightchains, and the light-chain constant region 1 (CL1) of theimmunoglobulin. It may further include the hinge region at theheavy-chain constant region. Hybrid Fc or hybrid Fc fragment issometimes referred to herein as “hFc.”

In addition, the Fc fragment of the present invention may be in the formof having native sugar chains, increased sugar chains compared to anative form or decreased sugar chains compared to the native form, ormay be in a deglycosylated form. The increase, decrease or removal ofthe immunoglobulin Fc sugar chains may be achieved by methods common inthe art, such as a chemical method, an enzymatic method and a geneticengineering method using a microorganism. The removal of sugar chainsfrom an Fc fragment results in a sharp decrease in binding affinity tothe C1q part of the first complement component C1 and a decrease or lossin antibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC), thereby not inducingunnecessary immune responses in vivo. In this regard, an immunoglobulinFc fragment in a deglycosylated or aglycosylated form may be, in somecases, more suitable to the object of the present invention as a drugcarrier.

As used herein, the term “deglycosylation” refers to that sugar moietiesare enzymatically removed from an Fc fragment, and the term“aglycosylation” means that an Fc fragment is produced in anunglycosylated form by a prokaryote, preferably E. coli.

The term “hybrid”, as used herein, means that sequences encoding two ormore immunoglobulin Fc fragments of different origin are present in asingle-chain immunoglobulin Fc fragment.

In an embodiment, the hybrid human Fc includes a hinge region, a CH2domain and a CH3 domain in an N-terminal to C-terminal direction,wherein the hinge region is an at least partial amino acid sequence of ahuman IgD hinge region or a human IgG1 hinge region; and the CH2 domainis of human IgG4 CH2 domain, a portion of which, at its N-terminalregion, is replaced by 4-37 amino acid residues of an N-terminal regionof a human IgG2 CH2 or human IgD CH2 domain. The hybrid human Fc can bejoined at its N-terminus to a C-terminus of a biologically activemolecule via a covalent bond.

In another embodiment, the biologically active molecule-hybrid Fc fusionpolypeptide may be represented by the following formula:N′—X—(Z1)_(p)-Y—Z2-Z3-Z4-C′, orN′—(Z1)_(p)-Y—Z2-Z3-Z4-(linker)_(q)-X—C′

wherein N′ is the N-terminus and C′ is the C-terminus of thepolypeptide; Z1 indicates an amino acid sequence including at least aC-terminal portion of the amino acid residues at positions 90 to 98 ofSEQ ID NO:11 or at least a portion of the amino acid residues atpositions 90-98 of SEQ ID NO:14; Y indicates an amino acid sequenceincluding at least a C-terminal portion of the amino acid residues atpositions 99 to 113 of SEQ ID NO:11 or at least a portion of the aminoacid residues at positions 99 to 162 of SEQ ID NO:14; Z2 indicates anamino acid sequence including at least an N-terminal portion of theamino acid residues at positions 111 to 147 of SEQ ID NO:12 or at leastan N-terminal portion of the amino acid residues at positions 163 to 199of SEQ ID NO:14; Z3 indicates an amino acid sequence including at leasta C-terminal portion of the amino acid residues at positions 118-223 ofSEQ ID NO:11, 114-219 of SEQ ID NO:12, 165-270 of SEQ ID NO: 24, or 115to 220 of SEQ ID NO:13; Z4 indicates an amino acid sequence including atleast an N-terminal portion of the amino acid residues at positions221-327 of SEQ ID NO:13; and p and q are each an integer of 0 or 1,wherein the total number of the amino acid residues for Z2 and Z3 isbetween 80 and 140, both inclusive, linker is a linker molecule, and Xis a biologically active molecule of interest.

In one embodiment, Z3-Z4 is an amino acid sequence selected from thegroup consisting of (i) a continuous amino acid sequence comprised ofthe C-terminal portion of the amino acid residues at positions 118 to223 of SEQ ID NO:11 and the N-terminal portion of the amino acidresidues at positions 224 to 330 of SEQ ID NO:11, (ii) a continuousamino acid sequence comprised of the C-terminal portion of the aminoacid residues at positions 114 to 219 of SEQ ID NO:12 and the N-terminalportion of the amino acid residues at positions 220 to 326 of SEQ IDNO:12, (iii) a continuous amino acid sequence comprised of theC-terminal portion of the amino acid residues at positions 165 to 270 ofSEQ ID NO: 24 and the N-terminal portion of the amino acid residues atpositions 271 to 377 of SEQ ID NO: 24, and (iv) a continuous amino acidsequence of the C-terminal portion of the amino acid residues atpositions 115 to 220 of SEQ ID NO: 13 and the N-terminal portion of theamino acid residues at positions 221 to 327 of SEQ ID NO:13.

The total number of amino acid residues of the polypeptide according toone embodiment of the present invention is from 154 to 288.

The polypeptides of the formula N′—X—(Z1)_(p)—Y—Z2-Z3-Z4-C′ andN′—(Z1)_(p)—Y—Z2-Z3-Z4-(linker)_(q)-X—C′ increases the circulatinghalf-life of the biologically active molecule X compared to acirculating half-life of X alone, when administered to a subject.

The linker may be derived from human albumin (CAA00606; SEQ ID NO: 25).The linker may comprise amino acid sequence 321 to 323, 318 to 325, 316to 328, 313 to 330, 311 to 333, or 306 to 338 of SEQ ID NO: 25.Alternatively, the linker may be a synthetic linker. The syntheticlinker may be a peptide composed of a total 10-20 residues of Gly andSer. In one embodiment, the Gly-Ser linker is GGGGSGGGGSGGGSG (SEQ IDNO: 32).

Z1 can comprise at least a portion of the CH1 domain of human IgG1 (SEQID NO:11) or IgD (SEQ ID NO:14). Z1 can comprise 5 to 9 or 7 to 9consecutive amino acid residues of the C-terminal region of the IgG1 CH1domain (positions 90-98 of SEQ ID NO: 11) or the C-terminal region ofthe IgD CH1 domain (positions 90-98 of SEQ ID NO: 14). In someembodiments, Z1 can be 5, 6, 7, 8 or 9 C-terminal amino acid residues ofthe IgG1 CH1 domain or IgD CH1 domain.

In some embodiments, Z1 is an amino acid sequence including amino acidresidues at positions 90 to 98 of SEQ ID NO: 11 or amino acid residuesat positions 90 to 98 of SEQ ID NO: 14. Z1 may be an amino acid sequenceconsisting of 5 to 9 amino acid residues at positions 90 to 98 of SEQ IDNO: 11 or amino acid residues at positions 90 to 98 of SEQ ID NO: 14. Z1also may be an amino acid sequence consisting of amino acid residues 90to 98 of SEQ ID NO: 11 or amino acid residues 90 to 98 of SEQ ID NO: 14.

Y can comprise at least a portion of the hinge region of human IgG1 orIgD. Y can comprise 5 or more, or 10 or more consecutive amino acidresidues of the C-terminal IgG1 hinge region (amino acid positions 99 to113 of SEQ ID NO: 11) or IgD hinge region (amino acid positions 99 to162 of SEQ ID NO: 14). In certain embodiments, Y can be an amino acidsequence including amino acid residues at positions 99 to 113 of SEQ IDNO: 11, amino acid residues at positions 158 to 162 of SEQ ID NO: 14,amino acid residues at positions 153 to 162 of SEQ ID NO: 14, amino acidresidues at positions 143 to 162 of SEQ ID NO: 14, amino acid residuesat positions 133 to 162 of SEQ ID NO: 14, or amino acid residues atpositions 99 to 162 of SEQ ID NO: 14.

Z2 can comprise 4 to 37, 6 to 30, 6 to 12, 6 to 8, 8 or 6 consecutiveamino acid residues of the N-terminal region of the human IgG2 CH2domain (the amino acid residues at positions 111 to 147 of SEQ ID NO:12) or the N-terminal region of the IgD CH2 domain (the amino acidresidues at positions 163 to 199 of SEQ ID NO: 14). In certainembodiments, Z2 can be 6 N-terminal amino aid residues of a human IgG2CH2 domain (amino acid residues 111-116 of SEQ ID NO: 12) or 8N-terminal amino acid residues of a human IgD CH2 domain (amino acidresidues 163-170 of SEQ ID NO:14).

The total number of amino acid residues of Z2 and Z3 can be between 90and 120, both inclusive, or 105 and 115, both inclusive.

Z4 can be an amino acid sequence including 90 or more, or 100 or moreconsecutive amino acid residues of the IgG4 CH3 domain (amino acidresidues at positions 224-330 of SEQ ID NO:11, 220-326 of SEQ ID NO:12,271-377 of SEQ ID NO: 24, or 221 to 327 of SEQ ID NO: 13). Z4 can be ofamino acid residues of more than 98% or 95% of the amino acid residuesof the human IgG1, IgG2, IgG3, or IgG4 CH3 domain. In one exemplaryembodiment, Z4 is an amino acid sequence comprising the entire aminoacid sequence of the human IgG CH3 domain. For example, Z4 is the aminoacid sequence of the human IgG4 CH3 domain, corresponding to amino acidresidues 341-447 of the human IgG4, as numbered according to the EUIndex, Kabat (which correspond to amino acid residues at positions221-327 of SEQ ID NO: 13).

In one embodiment, Y can be an amino acid sequence including at least aportion of the C-terminal amino acid residues of the human IgG1 hingeregion (amino acid residues 99-113 of SEQ ID NO: 11), p can be 1 or 0,Z2 can be an amino acid sequence including at least a portion of theN-terminal region of the human IgG2 CH2 (amino acid residues atpositions 111-147 of SEQ ID NO: 12), and Z3 can be an amino acidsequence including at least a portion of the C-terminal region of any ofthe human IgG subclasses (amino acid residues at positions 118-223 ofSEQ ID NO:11, 114-219 of SEQ ID NO:12, 165-270 of SEQ ID NO: 24, or 115to 220 of SEQ ID NO: 13). In this embodiment, when p is 1, Z1 can be anamino acid sequence including at least a portion of the C-terminalregion of the human IgG1 CH1 domain (amino acid residues at positions 90to 98 of SEQ ID NO: 11). For example, Z1 can be the amino acid residues90 to 98 of SEQ ID NO:11.

In further embodiments, Z3 can be 73 to 106 consecutive amino acidresidues of the C-terminal region of the human IgG4 CH2 domain(positions 115-220 of SEQ ID NO: 13), of the human IgG1 CH2 domain(positions 118-223 of SEQ ID NO:11), of the human IgG2 CH2 domain(positions 114-219 of SEQ ID NO:12), of the human IgG3 CH2 domain(positions 165-270 of SEQ ID NO: 24), and the total number of the aminoacid residues of Z2 and Z3 can be 110. For example, Z2 can be an aminoacid sequence of the amino acid residues at positions 111-116 of SEQ IDNO: 12, and Z3 can be an amino acid sequence of the amino acid residuesat positions 117 to 220 of SEQ ID NO: 13.

In another embodiment, Y can be an amino acid sequence including atleast a portion of the C-terminal region of the human IgD hinge region(amino acid residues at positions 99 to 162 of SEQ ID NO: 14), p can be1 or 0 (zero), Z2 can be an amino acid sequence including at least aportion of the N-terminal region of the human IgD CH2 domain (amino acidresidues at positions 163 to 199 of SEQ ID NO: 14), and Z3 can be anamino acid sequence including at least a portion of the C-terminalregion of the human IgG4 CH2 domain (amino acid residues at positions121 to 220 of SEQ ID NO: 13). For example, Y can be the amino acidresidues at positions 158 to 162, 133 to 162, or 99 to 162 of SEQ IDNO:14, Z2 can be the amino acid residues at positions 163 to 170 of SEQID NO:14, and Z3 can be amino acid residues at positions 121-220 of SEQID NO:13.

In this embodiment, when p is 1, Z1 can be an amino acid sequenceincluding the C-terminal region of the human IgD CH1 domain (amino acidresidues at positions 90 to 98 of SEQ ID NO: 14). For example, Z1 can beamino acid residues 90 to 98 of SEQ ID NO:14.

In this embodiment, Y can be 20 consecutive amino acid residues or more,30 consecutive amino acid residues or more, 40 consecutive amino acidresidues or more, 50 consecutive amino acid residues or more, or 60consecutive amino acid residues or more of the C-terminal side of thehuman IgD hinge region (amino acid residues at positions 99-162 of SEQID NO: 14). Z3 can comprise 71 to 100 consecutive amino acid residues ofthe C-terminal side of the amino acid residues at positions 121-220 ofSEQ ID NO: 13. The total number of the amino acid residues for Z2 and Z3can be 108.

Table 1 shows amino acid sequences of the fragments of human IgG1, IgG2,IgG3 and IgD useful in the construction of the hFcs according to theembodiments of the present invention.

TABLE 1 Acceptable Sequence of the longest fragments Location LocationhFc range of the within the acceptable range, in an in the in the EUdomain Ig fragments N-terminal to C-terminal direction SEQ ID NO: SEQ IDindex * CH1 (Z1) 5-9 C-terminal amino acid residues of IgG1 CH1

11 90-98 207-215 5-9 C-terminal amino acid residues of IgD CH1

14 90-98 Not Available Hinge (Y) 5-15 C-terminal amino acid residues ofIgG1 hinge region

11  99-113 216-230 5-64 C-terminal amino acid residues of IgD hingeregion

14  99-162 Not Available CH2, N-terminal side (Z2) 4-37 N-terminal aminoacid residues of IgG2 CH2

12 111-147 231-267 4-37 N-terminal amino acid residues of IgD CH2 domain

14 163-199 Not Available CH2, C-terminal side (Z3) + CH3 (Z4) 71-106C-terminal amino acid residues of IgG4 CH2 +

+ 13 115-220 + 221-327 235-340 + 341-447 80-107 N-terminal amino acidresidues of IgG4 CH3 domain

71-106 C-terminal amino acid residues of IgG3 CH2 +

+ 24 165-270 + 271-377 235-340 + 341-447 80-107 N-terminal amino acidresidues of IgG3 CH3 domain

71-106 C-terminal amino acid residues of IgG2 CH2 +

+ 12 114-219 + 220-326 234-340 + 341-447 80-107 N-terminal amino acidresidues of IgG2 CH3 domain

71-106 C-terminal amino acid residues of IgG1 CH2 +

+ 11 118-223 + 224-330 235-340 + 341-447 80-107 N-terminal amino acidresidues of IgG1 CH3 domain

*EU index is described in “Sequences of Proteins of ImmunologicalInterest, 5th Edition, United States Department of Health and HumanServices.” **The shaded region in each of the amino acid sequenceindicates the shortest fragments of the acceptable amino acid residueranges.

In an embodiment, the present invention provides a hybrid Fc which isone of hFc-1, hFc-2, hFc-3, hFc-4, hFc-5, or hFc-6, as shown in FIGS. 1and 2, or thFc-1 or thFc-2 as shown in FIGS. 3 and 4. Although FIGS. 1and 3 depict double chain Fcs, the present invention encompasses singlechain hybrid Fc molecules. Amino acid sequences of hFc-1 to hFc-6 areshown in SEQ ID NOs: 18-23, respectively and amino acid sequences ofthFc-1 and thFc-2 are shown in SEQ ID NO: 28 and SEQ ID NO: 29,respectively. The present invention also encompasses a polynucleotidemolecule coding for the hybrid Fc. They include, but are not limited to,a polynucleotide sequence as shown as SEQ ID NO: 1 (hFc-1), SEQ ID NO: 2(hFc-2), SEQ ID NO: 3 (hFc-3), SEQ ID NO: 4 (hFc-4), SEQ ID NO: 5(hFc-5), SEQ ID NO: 6 (hFc-6), SEQ ID NO: 26 (thFc-1) and SEQ ID NO: 27(thFc-2).

The amino acid sequences of human immunoglobulins are known in the artand they are deposited with a publicly accessible depository. Forexample, amino acid sequences of human IgG1 constant region, human IgG2constant region, human IgG3 constant region, human IgG4 constant region,and human IgD constant region are available at CAA75032, CAC20455,CAC20456, AAH25985 and P01880, respectively. These sequences arereproduced as SEQ ID NO: 11, 12, 24, 13 and 14, respectively.

A biologically active molecule X may be a soluble protein. It mayinclude, but is not limited to, a hormone, cytokine, growth factor,co-stimulatory molecule, hormone receptor, cytokine receptor, growthfactor receptor, or short peptide. For example, X may be an EPO, p40,G-CSF, TNF receptor or variants/fragments thereof. X may be a GM-CSF,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-10 receptor,TGF-beta, TGF-beta receptor, IL-17, IL-17 receptor, Factor VII, CXCL-11,FSH, human growth hormone, bone morphogenetic protein-1, CTLA4, PD-1,GLP-1, betacellulin, OPG, RNAK, interferon-alpha, interferon-beta ortheir variants/fragments. It also may include, but is not limited to, aFab region of an antibody. The biologically active molecule also may bea secreted protein. In one embodiment, the biologically active moleculedoes not belong to the immunoglobulin family.

The term “variant” refers to a polynucleotide or nucleic acid differingfrom a reference nucleic acid or polypeptide, but retaining essentialproperties thereof. Generally, variants are overall closely similar,and, in many regions, identical to the reference nucleic acid orpolypeptide. Also, the term “variant” refers to a biologically activeportion of a biologically active molecule drug, and retaining at leastone functional and/or therapeutic property thereof as describedelsewhere herein or otherwise known in the art. Generally, variants areoverall very similar, and, in many regions, identical to the amino acidsequence of the biologically active polypeptide of interest.

The present invention also provides proteins which comprise, oralternatively consist of, an amino acid sequence which is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example,the amino acid sequence of the polypeptides, as shown in SEQ ID NOs:18-23 and 28-29. Fragments of these polypeptides are also provided.Further polypeptides encompassed by the invention are polypeptidesencoded by polynucleotides which hybridize to the complement of anucleic acid molecule encoding the polypeptides of the invention understringent hybridization conditions (e.g., hybridization to filter boundDNA in 6× Sodium chloride/Sodium citrate (SSC) at about 45° C. followedby one or more washes in 0.2×SSC, 0.1% SDS at about 50-65° C.), underhighly stringent conditions (e.g., hybridization to filter bound DNA in6× sodium chloride/Sodium citrate (SSC) at about 45° C., followed by oneor more washes in 0.1×SSC, 0.2% SDS at about 68° C.), or under otherstringent hybridization conditions which are known to those of skill inthe art (see, for example, Ausubel, F. M. et al., eds., 1989 Currentprotocol in Molecular Biology, Green publishing associates, Inc., andJohn Wiley & Sons Inc., New York, at pages 6.3.1 6.3.6 and 2.10.3).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence, it is intended that theamino acid sequence of the subject polypeptide is identical to the querysequence except that the subject polypeptide sequence may include up tofive amino acid alterations per each 100 amino acids of the query aminoacid sequence. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a query amino acid sequence, upto 5% of the amino acid residues in the subject sequence may beinserted, deleted, or substituted with another amino acid. Thesealterations of the reference sequence may occur at the amino- orcarboxy-terminal positions of the reference amino acid sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence of an albumin fusion protein of the invention or afragment thereof, can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci. 6:237 245 (1990)). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of the globalsequence alignment is expressed as percent identity. Preferredparameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization GroupLength=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5,Gap Size Penalty=0.05, Window Size=500 or the length of the subjectamino acid sequence, whichever is shorter.

The variant will usually have at least 75% (preferably at least about80%, 90%, 95% or 99%) sequence identity with a length of normal HA orTherapeutic protein which is the same length as the variant. Homology oridentity at the nucleotide or amino acid sequence level is determined byBLAST (Basic Local Alignment Search Tool) analysis using the algorithmemployed by the programs blastp, blastn, blastx, tblastn and tblastx(Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264 2268 (1990) andAltschul, J. Mol. Evol. 36: 290 300 (1993), fully incorporated byreference) which are tailored for sequence similarity searching.

The polynucleotide variants of the invention may contain alterations inthe coding regions, non-coding regions, or both. Especially preferredare polynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, polypeptide variants in which less than 50, lessthan 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10,1-5, or 1-2 amino acids are substituted, deleted, added in anycombination are also preferred. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host, such as, yeast or E. coli).

For the purpose of constructing various Fc fusion proteins such asEPO-Fc fusion construct, G-CSF-Fc fusion construct, or human p40-Fcfusion construct, amino acid sequences of the human EPO, human G-CSF,human p40, and human TNF receptor are available from NP_(—)000790 (SEQID NO: 15), CAA27291(SEQ ID NO: 16), AAG32620 (SEQ ID NO: 17), andNP_(—)001057 (SEQ ID NO: 31), respectively. In one embodiment, amodified human p40 wherein amino acid residue Asn at position 303 isreplaced with Gln is connected to the polypeptide.

According to another aspect of the present invention, a whole antibodycontaining the engineered Fc region is provided. The term “antibody” asused herein includes whole antibodies and antibody fragments thatinclude at least two of CH1, hinge region, CH2 or CH3. Whole monoclonalantibodies are preferred. The heavy chain variable region of theantibody is selected for its binding specificity and can be of any type,such as, for example, non-human, humanized or fully human. Where theheavy chain variable region of the antibody is non-human (such as, forexample, murine) and is combined recombinantly with an engineered Fcregion in accordance with this disclosure, the resulting recombinantantibody is referred to as a chimeric antibody. Where the heavy chainvariable region of the antibody is humanized and is combinedrecombinantly with an engineered Fc region in accordance with thisdisclosure, the resulting recombinant antibody is referred to as ahumanized antibody. Where the heavy chain variable region of theantibody is human and is combined recombinantly with an engineered Fcregion in accordance with this disclosure, the resulting recombinantantibody is referred to as a fully human antibody. For example, thevariable region of the heavy chain is humanized and includes humanframework regions and non-human (in this case murine) complementarydetermining regions (CDRs). It should be understood that the frameworkregions can be derived from one source or more than one source and thatthe CDRs can be derived from one source or more than one source. Methodsfor humanization of antibodies are known to those skilled in the art andare known in the art.

The light chain of the antibody can be human, non-human or humanized. Inthe embodiment shown in Figure I B, the light chain is humanized andincludes human framework regions, non-human (in this case murine) CDRsand a human constant region. It should be understood that the frameworkregions can be derived from one source or more than one source and thatthe CDRs can be derived from one source or more than one source.

The antibody containing the engineered Fc region is selected based onits ability to bind to a cell surface molecule or a soluble moleculethat binds to a cell surface molecule. Thus, for example, the antibodycan be selected based on its ability to bind cell surface molecules suchas cytokine receptors (e.g., IL-2R, TNF-aR, IL-15R, etc.); adhesionmolecules (e.g., E-selectin, P-selectin, L-selectin, VCAM, ICAM, etc.);cell differentiation or activation antigens (e.g., CD3, CD4, CD8, CD20,CD25, CD40, etc.), and others. Alternatively, the antibody can beselected based on its ability to bind a soluble molecule that binds tocell surface molecules. Such soluble molecules include, but are notlimited to, cytokines and chemokines (e.g., interleukin-1 (IL-1), IL-2,IL-3, IL-5, IL-6, etc.); growth factors (e.g., EGF, PGDF, GM-CSF, HGF,IGF, BMP-1, etc.); molecules inducing cell differentiation (e.g., EPO,TPO, SCF, PTN, etc.), and others.

In general, the construction of the antibodies disclosed herein isachieved by, using recognized manipulations utilized in geneticengineering technology. For example, techniques for isolating DNA,making and selecting vectors for expressing the DNA, purifying andanalyzing nucleic acids, specific methods for making recombinant vectorDNA, cleaving DNA with restriction enzymes, ligating DNA, introducingDNA including vector DNA into host cells by stable or transient means,culturing the host cells in selective or non-selective media to selectand maintain cells that express DNA, are generally known in the field.

The monoclonal antibodies disclosed herein may be derived using thehybridoma method, which is known in the art, or other recombinant DNAmethods well known in the art. In the hybridoma method, a mouse or otherappropriate host animal is immunized with DNA, peptide or protein whichelicits the production of antibodies by the lymphocytes.

Alternatively, lymphocytes may be immunized in vitro. The lymphocytesproduced in response to the antigen are then are fused with myelomacells using a suitable fusing agent, such as polyethylene glycol, toform a hybridoma cell. The hybridoma cells are then seeded and grown ina suitable culture medium that preferably contains one or moresubstances that inhibit the growth or survival of the unfused, parentalmyeloma cells. Preferred myeloma cells are those that fuse efficiently,support stable production of antibody by the selected antibody-producingcells, and are not sensitive to a medium such as HAT medium (SigmaChemical Company, St. Louis, Mo., Catalog No. H-0262).

The antibodies containing the engineered Fc region can also be used asseparately administered compositions given in conjunction withtherapeutic agents. For diagnostic purposes, the antibodies may eitherbe labeled or unlabeled.

Unlabeled antibodies can be used in combination with other labeledantibodies (second antibodies) that are reactive with the engineeredantibody, such as antibodies specific for human immunoglobulin constantregions. Alternatively, the antibodies can be directly labeled. A widevariety of labels may be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors,ligands (particularly haptens), etc. Numerous types of immunoassays areavailable and are well known to those skilled in the art.

According to one embodiment, the present invention provides a method ofproducing the fusion protein, which method comprises: (i) introducing aDNA molecule coding for the fusion protein into a mammalian host cell,(ii) growing the cell under conditions the fusion protein is expressedin its growth medium; and (iii) harvesting the produced fusion protein.

In another exemplary embodiment, there is provided a pharmaceuticalcomposition comprising the fusion protein or an antibody molecule or anantibody fragment described above. It also provides a method of treatingor preventing certain symptoms by administering the pharmaceuticalcomposition. For example, a method is provided, which (i) reduces thesymptoms of/preventing/treating an autoimmune disease, (ii) inhibitsrejection of a graft, (iii) treats/prevents endotoxin-induced shock,comprising administering a therapeutically effective amount of thefusion protein of the hybrid Fc and a p40 protein or itsvariants/fragments.

The composition may comprises a pharmaceutical carrier. A pharmaceuticalcarrier can be any compatible, non-toxic substance suitable for deliveryof the antibodies to the patient. Sterile water, alcohol, fats, waxes,and inert solids may be included in the carrier. Pharmaceuticallyaccepted adjuvants (buffering agents, dispersing agent) may also beincorporated into the pharmaceutical composition.

The antibody compositions may be administered to a subject in a varietyof ways. For example, the pharmaceutical compositions may beadministered parenterally, e.g., subcutaneously, intramuscularly orintravenously. These compositions may be sterilized by conventional,well known sterilization techniques. The compositions may containpharmaceutical acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. The concentration of the fusion protein, antibody, orantibody fragment in these formulations can vary widely, e.g., from lessthan about 0.5%, usually at or at least about 1% to as much as 15 or 20%by weight and will be selected primarily based on fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected.

The present invention also provides an isolated nucleic acid moleculewhich encodes for the fusion protein, and an expression vector carryingthe nucleic acid molecule. Such nucleic acid may be directly deliveredto a subject which needs a polypeptide encoded by the nucleic acid.Alternatively, the polynucleotide is produced by expressing the nucleicacid in a medium and then administered to a subject.

The term “peptide,” “polypeptide” or “protein” refers to molecules of 2to 40 amino acids, with molecules of 3 to 20 amino acids preferred andthose of 6 to 15 amino acids most preferred. Exemplary peptides may berandomly generated by any of the methods cited above, carried in apeptide library (e.g., a phage display library), or derived by digestionof proteins.

The term “drug”, as used herein, refers to a substance displayingtherapeutic activity when administered to humans or animals, andexamples of the drug include, but are not limited to, polypeptides,compounds, extracts and nucleic acids. Preferred is a polypeptide drug.

The terms “physiologically active polypeptide,” “biologically activemolecule,” “physiologically active protein,” “active polypeptide,”“polypeptide drug,” and “protein drug”, as used herein, areinterchangeable in their meanings, and are featured in that they are ina physiologically active form exhibiting various in vivo physiologicalfunctions.

The polypeptide drug has a disadvantage of being unable to sustainphysiological action for a long period of time due to its property ofbeing easily denatured or degraded by proteolytic enzymes present in thebody. However, when the polypeptide drug is joined (or coupled) to theimmunoglobulin Fc fragments according to embodiments of the presentinvention to form a fusion protein, the drug has increased structuralstability and serum half-life. Also, the polypeptide joined to the Fcfragment has a much smaller decrease in physiological activity thanother known polypeptide drug formulations. Therefore, compared to the invivo bioavailability of conventional polypeptide drugs, the fusedpolypeptide comprising the polypeptide drug and the Fc fragment, or aconjugate of the polypeptide drug and the Fc fragment according to thepresent invention is characterized by having markedly improved in vivobioavailability. This is also clearly described through embodiments ofthe present invention. That is, when joined to the Fc fragment of thepresent invention, IFN-α, G-CSF, EPO, p40, TNF receptor, and otherprotein drugs displayed an increase in vivo bioavailability compared totheir native forms or other conventional fused forms.

It is understood that the present invention exploits conventionalrecombinant DNA methodologies for generating the Fc fusion proteins,antibodies containing engineered Fc region according to the presentinvention and antibody fragments useful in the practice of theinvention. The Fc fusion constructs preferably are generated at the DNAlevel, and the resulting DNAs integrated into expression vectors, andexpressed to produce the fusion proteins, antibody or antibody fragmentof the invention.

As used herein, the term “vector” is understood to mean any nucleic acidincluding a nucleotide sequence competent to be incorporated into a hostcell and to be recombined with and integrated into the host cell genome,or to replicate autonomously as an episome. Such vectors include linearnucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectorsand the like. Non-limiting examples of a viral vector include aretrovirus, an adenovirus and an adeno-associated virus. As used herein,the term “gene expression” or “expression” of a target protein, isunderstood to mean the transcription of a DNA sequence, translation ofthe mRNA transcript, and secretion of an Fc fusion protein product orantibody or antibody fragment.

A useful expression vector is RcCMV (Invitrogen, Carlsbad) or variantsthereof. The useful expression vector should carry human cytomegalovirus(CMV) promoter to promote constitute transcription of the interest genein mammalian cells and carry bovine growth hormone polyadenylationsignal sequence to increase steady state level of RNA aftertranscription. In an embodiment of the present invention, the expressionvector is pAD11, which is a modified vector of RcCMV. Examples of theexpression vector carrying a nucleotide sequence coding for abiologically active molecule drug may include, not is limited to, pAD11EPO-hFc-1, pAD11 G-CSF-hFc-1, pAD11 p40N303Q-hFc-1, pAD11 EPO-hFc-6,pAD11 G-CSF-hFc-6, pAD11 p40N303Q-hFc-6, pAD11 EPO-hFc-5, pAD11G-CSF-hFc-5, pAD11 p40N303Q-hFc-5 or pAD11 TNFR-hFc-5, as described inmore detail in Examples.

An appropriate host cell can be transformed or transfected with the DNAsequence of the invention, and utilized for the expression and/orsecretion of the target protein. Currently preferred host cells for usein the invention include immortal hybridoma cells, NS/0 myeloma cells,293 cells, Chinese hamster ovary cells, HeLa cells, and COS cells.

One expression system that has been used to produce high levelexpression of fusion proteins or antibody or antibody fragment inmammalian cells is a DNA construct encoding, in the 5′ to 3′ direction,a secretion cassette, including a signal sequence and an immunoglobulinFc region, and a target protein such as p40, EPO, G-CSF, TNF receptor.Several target proteins have been expressed successfully in such asystem and include, for example, IL2, CD26, Tat, Rev, OSF-2, ss; IG-H3,IgE Receptor, PSMA, and gp120. These expression constructs are disclosedin U.S. Pat. Nos. 5,541,087 and 5,726,044 to Lo et al., contents ofwhich are incorporated herein by reference.

The fusion proteins or antibody molecule or antibody fragments of theinvention may or may not be include a signal sequence when expressed. Asused herein, the term “signal sequence” is understood to mean a segmentwhich directs the secretion of the biologically active molecule drug;fusion protein and thereafter is cleaved following translation in thehost cell. The signal sequence of the invention is a polynucleotidewhich encodes an amino acid sequence which initiates transport of aprotein across the membrane of the endoplasmic reticulum. Signalsequences which are useful in the invention include antibody light chainsignal sequences, e.g., antibody 14.18 (Gillies et al., J. Immunol.Meth. 1989. 125:191-202), antibody heavy chain signal sequences, e.g.,the MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature1980. 286: 676-683), and any other signal sequences which are known inthe art (see, e.g., Watson et al., Nucleic Acids Research 1984.12:5145-5164).

Signal sequences have been well characterized in the art and are knowntypically to contain 16 to 30 amino acid residues, and may containgreater or fewer amino acid residues. A typical signal peptide consistsof three regions: a basic N-terminal region, a central hydrophobicregion, and a more polar C-terminal region. The central hydrophobicregion contains 4 to 12 hydrophobic residues that anchor the signalpeptide across the membrane lipid bilayer during transport of thenascent polypeptide. Following initiation, the signal peptide is usuallycleaved within the lumen of the endoplasmic reticulum by cellularenzymes known as signal peptidases. Potential cleavage sites of thesignal peptide generally follow the “(−3, −1) rule.” Thus a typicalsignal peptide has small, neutral amino acid residues in positions −1and −3 and lacks proline residues in this region.

The signal peptidase will cleave such a signal peptide between the −1and +1 amino acids. Thus, the signal sequence may be cleaved from theamino-terminus of the fusion protein during secretion. This results inthe secretion of an Fc fusion protein consisting of the immunoglobulinFc region and the target protein. A detailed discussion of signalpeptide sequences is provided by von Heijne (1986) Nucleic Acids Res.14:4683.

As would be apparent to one of skill in the art, the suitability of aparticular signal sequence for use in the secretion cassette may requiresome routine experimentation.

Such experimentation will include determining the ability of the signalsequence to direct the secretion of an Fc fusion protein and also adetermination of the optimal configuration, genomic or cDNA, of thesequence to be used in order to achieve efficient secretion of Fc fusionproteins. Additionally, one skilled in the art is capable of creating asynthetic signal peptide following the rules presented by von Heijne(1986), and testing for the efficacy of such a synthetic signal sequenceby routine experimentation. A signal sequence can also be referred to asa “signal peptide,” “leader sequence,” or “leader peptides.”

The fusion of the signal sequence and the immunoglobulin Fc region issometimes referred to as secretion cassette. An exemplary secretioncassette useful in the practice of the invention is a polynucleotideencoding, in a 5′ to 3′ direction, a signal sequence of animmunoglobulin light chain gene and an Fcy1 region of the humanimmunoglobulin y1 gene. The Fcy1 region of the immunoglobulin Fcy1 genepreferably includes at least a portion of the immunoglobulin hingedomain and at least the CH3 domain, or more preferably at least aportion of the hinge domain, the CH2 domain and the CH3 domain. As usedherein, the “portion” of the immunoglobulin hinge region is understoodto mean a portion of the immunoglobulin hinge that contains at leastone, preferably two cysteine residues capable of forming interchaindisulfide bonds. The DNA encoding the secretion cassette can be in itsgenomic configuration or its cDNA configuration. Under certaincircumstances, it may be advantageous to produce the Fc region fromhuman immunoglobulin Fcy2 heavy chain sequences. Although Fc fusionsbased on human immunoglobulin y1 and y2 sequences behave similarly inmice, the Fc fusions based on the y2 sequences can display superiorpharmacokinetics in humans.

In another embodiment, the DNA sequence encodes a proteolytic cleavagesite interposed between the secretion cassette and the target protein. Acleavage site provides for the proteolytic cleavage of the encodedfusion protein thus separating the Fc domain from the target protein. Asused herein, “proteolytic cleavage site” is understood to mean aminoacid sequences which are preferentially cleaved by a proteolytic enzymeor other proteolytic cleavage agents. Useful proteolytic cleavage sitesinclude amino acids sequences which are recognized by proteolyticenzymes such as trypsin, plasmin or enterokinase K. Many cleavagesite/cleavage agent pairs are known (see, for example, U.S. Pat. No.5,726,044).

Further, substitution or deletion of constructs of these constantregions, in which one or more amino acid residues of the constant regiondomains are substituted or deleted also would be useful. One examplewould be to introduce amino acid substitutions in the upper CH2 regionto create an Fc variant with reduced affinity for Fc receptors (Cole etal. (1997) J. Immunol. 159: 3613). One of ordinary skill in the art canprepare such constructs using well known molecular biology techniques.

Non-limiting examples of protein drugs capable of being conjugated tothe immunoglobulin Fc fragment of the present invention include humangrowth hormone, bone morphogenetic protein-1 (BMP-1), growth hormonereleasing hormone, growth hormone releasing peptide, interferons andinterferon receptors (e.g., interferon-α, -β and -γ, water-soluble typeI interferon receptor, etc.), granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),glucagon-like peptides (e.g., GLP-1, etc.), G-protein-coupled receptor,interleukins (e.g., interleukin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10,-11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24,-25, -26, -27, -28, -29, -30, etc.) and interleukin receptors (e.g.,IL-1 receptor, IL-4 receptor, etc.), enzymes (e.g., glucocerebrosidase,iduronate-2-sulfatase, alpha-galactosidase-A, agalsidase alpha and beta,alpha-L-iduronidase, butyrylcholinesterase, chitinase, glutamatedecarboxylase, imiglucerase, lipase, uricase, platelet-activating factoracetylhydrolase, neutral endopeptidase, myeloperoxidase, etc.),interleukin and cytokine binding proteins (e.g., IL-18 bp, TNF-bindingprotein, etc.), macrophage activating factor, macrophage peptide, B cellfactor, T cell factor, protein A, allergy inhibitor, cell necrosisglycoproteins, immunotoxin, lymphotoxin, tumor necrosis factor, tumorsuppressors, metastasis growth factor, alpha-1 antitrypsin, albumin,alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylatederythropoietin, angiopoietins; hemoglobin, thrombin, thrombin receptoractivating peptide, thrombomodulin, factor VII, factor VIIa, factorVIII, factor IX, factor XIII, plasminogen activating factor,fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C,C-reactive protein, renin inhibitor, collagenase inhibitor, superoxidedismutase, leptin, platelet-derived growth factor, epithelial growthfactor, epidermal growth factor, angiostatin, angiotensin, bone growthfactor, bone stimulating protein, calcitonin, insulin, atriopeptin,cartilage inducing factor, elcatonin, connective tissue activatingfactor, tissue factor pathway inhibitor, follicle stimulating hormone,luteinizing hormone, luteinizing hormone releasing hormone, nerve growthfactors (e.g., nerve growth factor, ciliary neurotrophic factor,axogenesis factor-1, brain-natriuretic peptide, glial derivedneurotrophic factor, netrin, neurophil inhibitor factor, neurotrophicfactor, neuturin, etc.), parathyroid hormone, relaxin, secretin,somatomedin, insulin-like growth factor, adrenocortical hormone,glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasingpeptide, corticotropin releasing factor, thyroid stimulating hormone,autotaxin, lactoferrin, myostatin, receptors (e.g., TNFR(P75),TNFR(P55), IL-1 receptor, VEGF receptor, B cell activating factorreceptor, etc.), receptor antagonists (e.g., IL1-Ra etc.), cell surfaceantigens (e.g., CD 2, 3, 4, 5, 7, 11a, 11b, 18, 19, 20, 23, 25, 33, 38,40, 45, 69, etc.), virus vaccine antigens, monoclonal antibodies,polyclonal antibodies, antibody fragments (e.g., scFv, Fab, Fab′,F(ab′)2 and Fd), and virus derived vaccine antigens. An antibodyfragment may be Fab, Fab′, F (ab′) 2, Fd or scFv, which is capable ofbinding to a specific antigen, and preferably Fab′. The Fab fragmentscontain the variable domain (VL) and constant domain (CL) of the lightchain and the variable domain (VH) and the first constant domain (CH1)of the heavy chain. The Fab′ fragments differ from the Fab fragments interms of adding several amino acid residues including one or morecysteine residues from the hinge region to the carboxyl terminus of theCH1 domain. The Fd fragments comprise only the VH and CH1 domain, andthe F (ab′) 2 fragments are produced as a pair of Fab′ fragments byeither disulfide bonding or a chemical reaction. The scFv (single-chainFv) fragments comprise the VL and VH domains that are linked to eachother by a peptide linker and thus are present in a single polypeptidechain.

In particular, preferred as biologically active molecules are thoserequiring frequent dosing upon administration to the body for therapy orprevention of diseases, which include human growth hormone, interferons(interferon-α, -β, -γ, etc.), granulocyte colony stimulating factor(G-CSF), erythropoietin (EPO), TFN receptor, p40, and antibodyfragments. In addition, certain derivatives are included in the scope ofthe biologically active molecules of the present invention as long asthey have function, structure, activity or stability substantiallyidentical to or improved compared over native forms of the biologicallyactive molecules. In the present invention, the most preferablepolypeptide drug is interferon-alpha.

In another aspect of the invention, IgG-Fc and IgG-CH fusion proteins,for example, are synthesized as monomers that can assemble to formdimers. Typically, the dimers are joined by disulfide bonds in the IgGHinge region. Conditioned media from cells secreting the IgG fusionproteins can contain mixtures of IgG fusion protein monomers and dimers.For use as human therapeutics it will be desirable to use homogeneouspopulations of either IgG fusion protein monomers or dimers, but notmixtures of the two forms.

Methods for obtaining essentially pure preparations of dimeric activepolypeptide-IgG fusion proteins are also provided. The methods aregenerally accomplished by obtaining a host cell capable of expressingthe IgG fusion protein, collecting the conditioned media, and purifyingthe dimeric fusion protein from monomeric fusion protein, aggregates andcontaminating proteins by column chromatography procedures. Suitablehost cells for expressing the IgG fusion proteins include yeast, insect,mammalian or other eukaryotic cells. In an embodiment, the host cell maybe a mammalian cell, particularly COS, CHO or BHK cells.

Novel fusion proteins of a polypeptide drug and a Fc fragment are alsoprovided. In one embodiment, a polypeptide drug such as EPO, p40, G-CSFor TNF receptor is joined directly to the hybrid Fc fragment without anintervening peptide linker. In another embodiment, the polypeptide drugis joined to each other through a peptide linker of 1 to 50 amino acids,and more preferably through a peptide linker of 1 to 7 amino acids.Particularly useful linkers for this purpose include an immunologicallyinactive peptide composed of Gly and Ser residues (eg. Gly Gly Ser GlyGly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser; SEQ ID NO: 32) orcomposed of amino acids at positions 282-314 of SEQ ID NO: 25 derived inhuman albumin.

In the case when a linker is used, the linker and a polypeptide drug maybe made in a certain direction. That is, the linker may be linked to theN-terminus, the C-terminus or a free group of the hybrid Fc fragment,and may also be linked to the N-terminus, the C-terminus or a free groupof the polypeptide drug. When the linker is a peptide linker, thelinkage may take place at a certain linking site.

When a polypeptide drug and a hybrid Fc is expressed separately and thenjoined to each other, the coupling may be performed using any of anumber of coupling agents known in the art. Non-limiting examples of thecoupling agents include 1,1-bis(diazoacetyl)-2-phenylethane,glutaradehyde, N-hydroxysuccinimide esters such as esters with4-azidosalicylic acid, imidoesters including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane.

The present invention also provides methods for the production ofpolypeptide drug-hybrid Fc fragment.

The present invention also provides methods for treating conditionsalleviated by the administration of a polypeptide drug. These methodsinclude administering to a mammal having the condition, which may or maynot be directly related to a disease of interest, an effective amount ofa polypeptide of the invention. For example, a nucleic acid, such as DNAor RNA, encoding a desired polypeptide drug-hybrid Fc fragment fusionprotein can be administered to a subject, preferably a mammal, as atherapeutic agent. Additionally, a cell containing a nucleic acidencoding a polypeptide drug-hybrid Fc fragment fusion protein can beadministered to a subject, preferably a mammal, as a therapeutic agent.Furthermore, a polypeptide drug-hybrid Fc fragment fusion construct canbe administered to a subject, preferably a mammal, as a therapeuticagent. Such chimeric polypeptide may be administered intravenously,subcutaneously, orally, buccally, sublingually, nasally, parenterally,rectally, vaginally or via a pulmonary route.

An EPO (including its variants/fragments)-fc fusion protein of thepresent invention may be useful in raising and maintaining hematocrit ina mammal.

p40 is a subunit of IL-12. IL-12 is a 75 kDa heterodimeric cytokine thathas several functions in vivo. For example, IL-12 stimulatesproliferation of activated T and NK cells and promotes Th1-type helpercell responses. IL-12 exerts its biological effects by binding to theIL-12 receptor on the plasma membrane of activated T and NK cells, andthe ability of IL-12 to bind to the IL-12 receptor has been attributedto the p40 subunit of IL-12. Therefore, a p40 (including itsvariants/fragments)-fc fusion protein of the present invention may beuseful in reducing the symptoms of/preventing/treating an autoimmunedisease, (ii) inhibiting rejection of a graft, or (iii)treating/preventing endotoxin-induced shock. Also, a p40 (including itsvariants/fragments)-fc fusion protein of the present invention may beuseful in treating/preventing/amelioration the symptoms of rheumatoidarthritis, ankylosing spondylitis, inflammatory bowl disease, multiplesclerosis or psoriasis. Variants and fragments are known in the art,including, not limited to, WO 97/20062, contents of which areincorporated herein as reference. One embodiment of p40 variantincludes, but is not limited to, p40 containing Asn303Gln substitution.

Granulocyte colony stimulating factor (G-CSF) is a protein that isessential for the proliferation and differentiation of granulocytes,particularly neutrophils. Granulocytes engulf and devour microbialinvaders and cell debris and thus are crucial to infection response.Chemotherapy destroys granulocytes and/or decrease the production ofgranulocytes. Therefore, a G-CSF (including its variants/fragments)-fcfusion protein of the present invention may be useful intreating/preventing/amelioration the symptoms of chemotherapy-inducedneutropenia myelosuppression after bone marrow transplantation, acuteleukemia, aplastic anemia, myelodysplastic syndrome, severe chronicneutropenias, or mobilization of peripheral blood progenitor cells fortransplantation.

The fusion proteins of the invention not only are useful as therapeuticagents, but one skilled in the art recognizes that the fusion proteinsare useful in the production of antibodies for diagnostic use. Likewise,appropriate administration of the DNA or RNA, e.g., in a vector or otherdelivery system for such uses, is included in methods of use of theinvention.

Compositions of the present invention may be administered by any routewhich is compatible with the particular molecules. It is contemplatedthat the compositions of the present invention may be provided to ananimal by any suitable means, directly (e.g., locally, as by injection,implantation or topical administration to a tissue locus) orsystemically (e.g., parenterally or orally). Where the composition is tobe provided parenterally, such as by intravenous, subcutaneous,ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal,intraorbital, intracerebral, intracranial, intraspinal,intraventricular, intrathecal, intracistemal, intracapsular, intranasalor by aerosol administration, the composition preferably includes partof an aqueous or physiologically compatible fluid suspension orsolution. Thus, the carrier or vehicle is physiologically acceptable sothat in addition to delivery of the desired composition to the patient,it does not otherwise adversely affect the patient's electrolyte and/orvolume balance. The fluid medium for the agent thus can include normalphysiologic saline.

The DNA constructs (or gene constructs) of the invention also can beused as a part of a gene therapy protocol to deliver nucleic acidsencoding a polypeptide drug or a fusion protein construct thereof.

The invention features expression vectors for in vivo transfection andexpression of a polypeptide drug of interest or a fusion proteinconstruct thereof in particular cell types so as to reconstitute orsupplement the function of the desired polypeptide drug. Expressionconstructs of the desired polypeptide drug, or fusion protein constructsthereof, may be administered in any biologically effective carrier, e.g.any formulation or composition capable of effectively delivering thedesired polypeptide drug-encoding gene or fusion protein constructthereof to cells in vivo.

Approaches include insertion of the subject gene in viral vectorsincluding recombinant retroviruses, adenovirus, adeno-associated virus,and herpes simplex virus-1, or recombinant bacterial or eukaryoticplasmids. Preferred dosages per administration of nucleic acids encodingthe fusion proteins of the invention are within the range of 0.1 mg-100mg for humans, more preferably 1 mg-10 mg, and most preferably 2 mg-10mg. It is contemplated that the optimal dosage and mode ofadministration may be determined by routine experimentation well withinthe level of skill in the art.

Preferred dosages of the fusion protein per administration are withinthe range of 0.1 mg-1,000 mg for humans, more preferably, 1 mg-100 mgand most preferably 5 mg-20 mg. It is contemplated that the optimaldosage, however, also depends upon the disease being treated and uponthe existence of side effects. However, optimal dosages may bedetermined using routine experimentation. Administration of the fusionprotein may be by periodic bolus injections, or by continuousintravenous, subcutaneous, or intraperitoneal administration from anexternal reservoir (for example, from an intravenous bag) or internal(for example, from a bioerodable implant).

Furthermore, it is contemplated that the fusion proteins of theinvention also may be administered to the intended recipient togetherwith a plurality of different biologically active molecules. It iscontemplated, however, that the optimal combination of fusion proteinand other molecules, modes of administration, dosages may be determinedby routine experimentation well within the level of skill in the art.

The invention is illustrated further by the following non-limitingexamples.

Example 1 Preparation of Expression Vectors for hFc-1, hFc-2, hFc-3,hFc-4, hFc-5, and hFc-6 Fusion Proteins

The hFc-1 includes 9 amino acids (90-98) of C-terminal IgG1 CH1 region,hinge region (99-113) of IgG1, 6 amino acids (111-116) of N-terminalIgG2 CH2 region, 103 amino acids (118-220) of IgG4 CH2 region, and 107amino acids (221-327) of IgG4 CH3 region (FIGS. 1 and 2). An amino acidsequence of hFc-1 is shown in SEQ ID NO: 18. To obtain codon-optimizednucleotides each coding for hFc-1 (SEQ ID NO: 1), human EPO (SEQ ID NO:7), human G-CSF (SEQ ID NO: 8) and human p40N303Q (a mutant derived fromsubstitution of Asn with Gln at the 303rd amino acid of human p40subunit) (a nucleotide sequence of p40N303Q is shown as SEQ ID NO: 9,and an amino acid sequence of human p40 is shown as SEQ ID NO: 17),respectively, these nucleotide molecules were synthesized by customservice of TOP Gene Technologies (Quebec, Canada) (www.topgenetech.com).To increase the level of protein expression, it is very helpful tooptimize codon usage of the gene. The pattern of codon usage differsbetween organisms. Some codons are used more frequently in one organismbut used rarely in another organism. This bias in codon usage has beenattributed to translational efficiency, the ability of the organism tosynthesize the encoded protein. To insert each fusion gene to anexpression vector, pAD11 (SEQ ID NO: 10), a EcoR I site was generated at5′ end of ATG sequence of EPO, G-CSF, and p40N303Q and Xba I site wasgenerated at 3′ end of the termination codon of hFc-1. The expressionvector pAD11 was obtained from RcCMV backbone (available fromInvitrogen, Carlsbad). pAD11, includes a promoter derived fromcytomegalovirus (CMV), poly (A) sequences derived from bovine growthhormone, globin intervening sequence (gIVS) derived from rabbit betaglobin (Mol Cell Biol, 1988 8: 4395) and etc. To make the pAD11 vector,there are several modifications from the RcCMV vector (Invitrogen). Aneomycin resistant region was removed by treatment with Xho I enzyme andgIVS was added at 3′ of CMV promoter region. In addition, a mousedihydrofolate reductase (DHFR) gene (Pubmed, NM 010049) was added at 5′of CMV promoter. The pAD11 vector was developed after many expressiontests in combination with several elements including them describedabove. In our unpublished result, pAD11 vector showed about 12-foldincrease in expression level, compared to RcCMV vector (Invitrogen). Tomake a junction site between 3′ end of EPO, G-CSF and p40N303Q and 5′end of hFc-1 in frame, a Nhe I site at 3′ end of the coding sequence ofEPO, G-CSF and p40N303Q and at 5′ end of the coding sequence of hFc-1was generated. After subcloning using each restriction enzymes site, thefinal expression vectors for hFc-1 fused with EPO, G-CSF or p40N303Qwere generated, and then designated as pAD11 EPO-hFc-1, pAD11G-CSF-hFc-1 and pAD11 p40N303Q-hFc-1, respectively.

Amino acid sequences of hFc-2, hFc-3, hFc-4, hFc-5 and hFc-6 are shownin SEQ ID NOs: 19-23, respectively. The hFc-6 includes 9 amino acids(90-98) of the C-terminal IgD CH1 domain, 64 amino acids of the hingeregion (99-162) of IgD, 8 amino acids (SHTQPLGV; 163-170) of theN-terminal IgD CH2 domain, 100 amino acids (121-220) of the IgG4 CH2domain, and 107 amino acids (221-327) of the IgG4 CH3 domain (FIGS. 1and 2). To obtain codon-optimized nucleotide molecule coding for hFc-6(SEQ ID NO: 6), the gene was synthesized by custom service of TOP GeneTechnologies (www.topgenetech.com). To make a fusion between 3′ end ofEPO, G-CSF, or p40N303Q and the 5′ end of hFc-6 in frame, the Nhe I site(gctagc: Ala-Ser) included in the N-terminal coding region (90 and 91amino acids) of hFc-6 was used. Also, to insert each hFc-6 fusion geneinto pAD 11 vector, a Xba I site was generated at the 3′ end of hFc-6gene. After subcloning using each restriction enzymes site, the finalexpression vectors for hFc-6 fused EPO, G-CSF and p40N303Q weregenerated, and then designated as pAD11 EPO-hFc-6, pAD11 G-CSF-hFc-6 andpAD11 p40N303Q-hFc-6, respectively. The hFc-2, hFc-3, hFc-4, and hFc-5have identical CH2 and CH3 regions, but they have different sizes of IgDhinge (FIGS. 1 and 2). The hFc-2 (SEQ ID NO: 19), hFc-3 (SEQ ID NO: 20),hFc-4 (SEQ ID NO: 21), and hFc-5 (SEQ ID NO: 22) includes 5 amino acids(158-162), 10 amino acids (153-162), 20 amino acids (143-162), 30 aminoacids (133-162) of C-terminal IgD hinge, respectively (FIGS. 1 and 2).To make the fusion genes between EPO, G-CSF, p40N303Q or TNFR (tumornecrosis factor receptor II) (SEQ ID NO: 30) and nucleic acid moleculescoding for these hFcs (SEQ ID NOs: 2-5), the minimal gene fragments intotal size of the fused genes were synthesized by custom service of TOPGene Technologies (www.topgenetech.com). The synthesized fragments ofeach EPO, G-CSF, p40N303Q or TNFR fused with a nucleotide moleculecoding for hinge and N-terminal CH2 region of each hFc-2, hFc-3, hFc-4,or hFc-5 include the sequences ranged from the entire EPO, G-CSF,p40N303Q or TNFR sequences to the identical enzyme site, BstE II site(GGTGACC) that is located at 138-140^(th) amino acid residues of CH2region in IgG4 (SEQ ID NO: 13). The subcloning vectors including severalgene fragments were cut with EcoR I and BstE II located at 5′ end and 3′end, respectively, and then ligated to the CH2-CH3 region of the hFc-6.Finally, the each fusion gene was subcloned to the pAD11 using EcoR Iand Xba I sites, and then designated as pAD11 EPO-hFc-2, pAD11EPO-hFc-3, pAD11 EPO-hFc-4, pAD11 EPO-hFc-5, pAD11 G-CSF-hFc-2, pAD11G-CSF-hFc-3, pAD11 G-CSF-hFc-4, pAD11 G-CSF-hFc-5, pAD11 p40N303Q-hFc-2,pAD11 p40N303Q-hFc-3, pAD11 p40N303Q-hFc-4, pAD11 p40N303Q-hFc-5 andpAD11 TNFR-hFc-5, respectively.

Example 2 Preparation of Expression Vectors for thFc-1 and thFc-2Coupled to IFN-13

The thFc-1 includes 23 amino acids (MDAMLRGLCCVLLLCGAVFVSPS) of signalsequence of human tissue plasminogen activator (tPA), 15 amino acids(99-113) of IgG1 hinge region, 6 amino acids (111-116) of N-terminalIgG2 CH2 region, 103 amino acids (118-220) of IgG4 CH2 region, and 107amino acids (221-327) of IgG4 CH3 region (FIG. 3). An amino acidsequence of thFc-1 is shown in SEQ ID NO: 28. The thFc-2 includes 23amino acids (MDAMLRGLCCVLLLCGAVFVSPS) of tPA signal sequence, 15 aminoacids (148-162) of IgD hinge region, 8 amino acids (163-170) ofN-terminal IgD CH2 region, 100 amino acids (121-220) of IgG4 CH2 region,and 107 amino acids (221-327) of IgG4 CH3 region (FIG. 3). An amino acidsequence of thFc-2 is shown in SEQ ID NO: 29. To obtain codon-optimizednucleotides coding for thFc-1 (SEQ ID NO: 26) or thFc-2 (SEQ ID NO: 27)coupled to the N-terminus of human IFN-beta deleted its signal sequence,these nucleotide molecules were synthesized by custom service of TOPGene Technologies (Quebec, Canada) (www.topgenetech.com). To insert eachfusion gene to an expression vector, pAD11 (SEQ ID NO: 10), a EcoR Isite was generated at 5′ end of thFc-1 or thFc-2 and Not I site wasgenerated at 3′ end of the termination codon of IFN-beta. Aftersubcloning using each restriction enzymes site, the final expressionvectors were designated as pAD11 thFc-1-AL(0)-IFN-beta and pAD11thFc-2-AL(0)-IFN-beta, respectively.

To make thFc coupled to IFN-beta via different sizes of albumin linkersor Gly-Ser linker, the gene fragments ranged from Pst I site of CH3region of thFc-1 coupled to IFN-beta deleted its signal sequence viadifferent sizes of albumin linkers (3aa, 8 aa, 13aa, 18aa, 23aa and33aa) or Gly-Ser linker (15aa) were synthesized by custom service of TOPGene Technologies (www.topgenetech.com) (FIG. 4). To insert 7 differentgene fragments to expression vectors, pAD11 thFc-1-AL(0)-IFN-beta andpAD11 thFc-2-AL(0)-IFN-beta, a Pst I site was generated at 5′ end ofthem and Not I site was generated at 3′ end of the termination codon ofIFN-beta. After subcloning using each restriction enzymes site, thefinal expression vectors were designated as pAD11 thFc-1-AL(1)-IFN-beta,pAD11 thFc-1-AL(2)-IFN-beta, pAD11 thFc-1-AL(3)-IFN-beta, pAD11thFc-1-AL(4)-IFN-beta, pAD11 thFc-1-AL(5)-IFN-beta, pAD11thFc-1-AL(6)-IFN-beta pAD11, thFc-1-GS-IFN-beta, pAD11thFc-2-AL(1)-IFN-beta, pAD11 thFc-2-AL(2)-IFN-beta, pAD11thFc-2-AL(3)-IFN-beta, pAD11 thFc-2-AL(4)-IFN-beta, pAD11thFc-2-AL(5)-IFN-beta, pAD11 thFc-2-AL(6)-IFN-beta pAD11, andthFc-2-GS-IFN-beta.

Example 3 Expression of Human EPO-hFcs, Human G-CSF-hFcs, Humanp40N303Q-hFcs, Human TNFR-hFc-5 and thFcs-IFN-Beta Proteins

COS-7 cells were used for expression test and cultured with DMEM media(Invitrogen, Carlsbad) supplemented with 10% fetal bovine serum(Hyclone, South Logan) and antibiotics (Invitrogen, Carlsbad). Thevectors encoding EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5,thFcs-IFN-beta were transfected to 5×10⁶ COS-7 cells using conventionalelectroporation methods. At 48 h after transfection, supernatants andcells were harvested. To check the expression of fusion protein fromeach vector, all the samples were used for ELISA assay with several kits(R&D system, Minneapolis, #DEP00 for EPO; Biosource, Camarillo,#KHC2032, for G-CSF; R&D system, Minneapolis, #DY1240 for p40N303Q; R&Dsystem, Minneapolis, #DRT200 for TNFR, PBL Biomedical Laboratories,#41410-1A for IFN-beta) and western blot analysis with anti-human IgGantibodies (Santa Cruz Biotechnology, Santa Cruz). As a result, all thevectors showed correct expression pattern in the supernatants and celllysates (data not shown).

Example 4 Purification of hFc-Fused Proteins

The CHO/DHFR^(−/−) cells (chinese hamster ovary cells, DG44, ATCC) werecultured with α-MEM (Invitrogen, Carlsbad), 10% dialyzed fetal bovineserum (JRH Biosciences, Kansas), HT supplement (Invitrogen, Carlsbad)and antibiotics (Invitrogen, Carlsbad). The expression vectors weretransfected to the CHO cells according to the conventional CaPO₄coprecipitation methods. At 48 h after transfection, the CHO cells weredetached from the plates and diluted at several folds (1/2, 1/5, 1/20,1/50, 1/100, 1/200, 1/500). The diluted cells were plated to 100 mmdishes and cultured with the media without HT supplement. Duringscreening process, the fresh media without HT supplement were suppliedto the cells without passage. The colonies were generated for 2-3 weeksafter plating and the individual colonies were moved to 48 well plates.The positive colonies were screened after ELISA assay for EPO, G-CSF,p40N303Q, and TNFR detections. Each colony that showed the highestexpression was cultured in a large scale (5 L) using serum free media(JRH Biosciences, Kansas). The serum-free supernatants harvested wereused for purification of each fusion protein. For purification, HiTraprecombinant protein A FF (Amersham biosciences, Piscataway) columns wereequilibrated with 20 mM Sodium phosphate (pH 7.0). The filteredsupernatants were added to the columns and eluted with 0.1M sodiumcitrate (pH 3.0). The eluted proteins were finally obtained afterdialysis with membrane (MWCO 12-14K, Spectrapor, Rancho Dominguez) morethan three times. All the concentration of protein samples wasdetermined by BCA kit (Pierce Biotechnology, Rockford) for themeasurement of the total protein and by ELISA kits for the measurementof EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5 and thFcs-IFN-beta.

Example 5 FcγRI and C1q Binding Assay

To investigate whether hFc-5-fused proteins bind to FcγRI and C1q,MabThera® (Rituximab, Roche), hIgG1 (Calbiochem, Cat#, 400120), Enbrel®(etanercept, Amgen), EPO-hFc-5, G-CSF-hFc-5 and p40N303Q-hFc-5 wereserially diluted (from 2 ug/ml to 16 ng/ml with 2-fold) and coated onthe 8 well strip (COSTAR, New York) overnight at 4° C. To make astandard curve, FcγRI (R&D, cat# BAF1257) or C1q (AbD serotech, Cat#.2221-5504) were also serially diluted (from 2 ug/ml to 32 ng/ml with2-fold) and coated on the 8 well strip (COSTAR, New York) overnight at4° C. After washing each strip of samples with washing buffer (PBScontaining 0.05% Tween) and blocking with 10% FBS in PBS for 1 hour atRT, FcγRI or C1 q were added into each well at 2 ug/ml followingincubation for 2 hours at room temperature (RT). All strips were washedwith washing buffer. For C1 q binding test, HRP conjugated anti-C1q (AbDserotech, cat#. 2221-5004P) was added into each well at 2.5 ug/mlfollowing 30 min incubation at RT under dark condition. For FcγRIbinding test, biotinylated anti-FcγRI (R&D, cat#. 1257-FC) was addedinto each well at 2 ug/ml following 1 hour incubation at RT. Afterwashing them with washing buffer, Streptavidine-HRP (BD, cat#. 554066)diluted with 3,000 fold was added into each strip following 30 minuteincubation at RT under dark condition. After washing the strips, TMBsolution (1:1 mixture of TMB Peroxidase substrate and Peroxidasesubstrate solution B, KPL, cat#. 50-76-01, cat#, 50-65-00) was added fordevelopment and 2N H₂SO₄ was added for stopping development. As shown inFIG. 6( a) and FIG. 6( b), MabThera®, Enbrel® and hIgG1 were shown to bewell bound to FcγRI and C1q, but EPO-hFc-5, G-CSF-hFc-5 andp40N303Q-hFc-5 were not.

Example 6 In Vitro Bioactivity of Purified hFc-Fused Proteins

To investigate the in vitro bioactivities of EPO-hFc proteins, humanF35E cell line was cultured in RPMI1640 media (Cambrex, Charles City)supplemented with 10% FBS, antibiotics and 5 IU/ml recombinant human EPO(DongA, Republic of Korea). Bioassays were set up by seeding 2×10⁴ cellsto test wells of a 96-well cell culture plate (Corning, Netherlands).The samples with serial dilutions (0, 0.064 mIU/ml to 25 IU/ml with5-fold) of EPO, EPO-hFc-1, EPO-hFc-5, EPO-hFc-6, EPO-IgG1 Fc or Aranesp®(darbepoetin alfa, Amgen) were added to the these wells and the plateswere incubated at 37° C. for 72 hours in a humidified 5% CO₂ incubator.According to the manufacturer's protocol, MTT assay was performed byusing cell growth colorimetric assay kit (Sigma-Aldrich. Korea). Thehuman F35E cell line showed a strong proliferative response to rEPO, asevidenced by a dose-dependent manner in cell number and absorbancevalues. As shown in FIG. 7( a), Aranesp® and EPO proteins coupled toIgG1 Fc or hFcs showed loss of biological activity, compared to EPOprotein. However, EPO-hFc-1, EPO-hFc-5 and EPO-hFc-6 showedsignificantly higher bioactivity than EPO-IgG1 Fc. In addition,EPO-hFc-5 and EPO-hFc-6 showed slightly higher bioactivity thanAranesp®, indicating that these hFc-fused proteins appear to be betterthan Aranesp® in terms of maintaining bioactivity of EPO protein.

To investigate the in vitro bioactivities of G-CSF-hFc protein, mousehematopoietic cell line, NFS-60 was cultured in RPMI1640 media (Cambrex,Charles City) supplemented with 10% FBS, antibiotics and 100 units/mlrecombinant mouse IL-3 (R&D system, Minneapolis). Bioassays were set upby seeding 2×10⁴ cells to wells of a 96-well cell culture plate(Corning, Netherlands). The samples with serial dilutions (ranged from 0to 10,000 pg/ml with 3-fold) of G-CSF-hFc-5 and Neulasta®(pegfilgrastim, Amgen) were added to these wells and the plates wereincubated at 37° C. for 72 hours in a humidified 5% CO₂ incubator.Protein samples were assayed in triplicate wells and this experiment wasperformed repeatedly for five times. At 72 hours after incubation, MTTassay was performed by using cell growth colorimetric assay kit(Sigma-Aldrich. Korea), according to the manufacturer's protocol. Asillustrated in FIG. 7( b), G-CSF-hFc-5 showed slightly higher in vitrobioactivity than Neulasta®.

To investigate the in vitro bioactivity of p40N303Q-hFc protein,peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritispatients were incubated with 2 ug/ml of anti-human CD3 antibody (R&Dsystem, # MAB100) with or without 10 ng/ml of human p40 (R&D system) orp40N303Q-hFc-5 in RPMI1640 media (Cambrex, Charles City) supplementedwith 10% FBS, and antibiotics. After day 6, the cells positive for CD4and IL-17 were measured by FACS analysis. As shown in FIG. 7( c),p40N303Q-hFc-5 showed stronger suppressive effect on the generation ofCD4⁺/IL-17⁺ cells than p40 protein, indicating the inhibitory functionof p40N303Q-hFc-5 on Th17 polarization.

To investigate the in vitro bioactivity of TNFR-hFc protein, murine L929cells were cultured in RPMI1640 media (Cambrex, Charles City)supplemented with 10% FBS and antibiotics. Cytopathic inhibition assaywas set up by seeding 3×10⁴ cells to wells of a 96-well cell cultureplate (Corning, Netherlands), then treated with 1 ng/ml of TNF-α. Thesamples with serial dilutions (ranged from 15.6 to 1,000 ng/ml with2-fold) of TNFR-hFc-5 and Enbrel® (etanercept, Amgen) were added tothese wells and the plates were incubated at 37° C. for 48 hours in ahumidified 5% CO₂ incubator. After incubation, MTT assay was performedby using cell growth colorimetric assay kit (Sigma-Aldrich, Korea),according to the manufacturer's protocol. As illustrated in FIG. 7( d),TNFR-hFc-5 showed slightly higher in vitro bioactivity than Enbrel®.

To investigate the in vitro bioactivities of thFc-1-AL(0)-IFN-beta andthFc-1-AL(3)-IFN-beta proteins, WISH cells (ATCC, CCL-25) were culturedin DMEM/F12 (Cambrex, Charles City) supplemented with 10% FBS andantibiotics. Cytopathic inhibition assay was set up by seeding 3×10⁴cells to wells of a 96-well cell culture plate (Corning, Netherlands),then treated with 1,500 PFU/well of VSV (ATCC, VR-158). The samples withserial dilutions (from 40 IU/ml with 2-fold) of recombinant IFN-beta(WHO standard, NIBSC 00/572), thFc-1-AL(0)-IFN-beta andthFc-1-AL(3)-IFN-beta proteins were added to these wells and the plateswere incubated at 37° C. for 48 hours in a humidified 5% CO₂ incubator.After incubation, MTT assay was performed by using cell growthcolorimetric assay kit (Sigma-Aldrich. Korea), according to themanufacturer's protocol. As illustrated in FIG. 7( e),thFc-1-AL(3)-IFN-beta showed about 20 fold higher in vitro bioactivitythan thFc-1-AL(0)-IFN-beta, indicating the important role of albuminliker to maintain the bioactivity of IFN-beta fused to Fc.

Example 7 In Vivo Half Life of Purified hFc-Fused Proteins

To compare the half life of EPO-hFc-1, EPO-hFc-5 and Aranesp®, fifteencynomolgus monkeys were treated with these proteins in a dose of 2,400IU/kg via single subcutaneous (SC) injection or a single intravenous(IV) injection. Blood samples of each monkey were obtained beforeinjection and at 1, 3, 6, 12, 24, 30, 48, 54, 72, 78, 96, 120, 168, 336,504, and 672 h post-injection. Blood samples were incubated at roomtemperature for 30 min to be clotted. After centrifugation at 3000 rpmfor 10 min, sera from each sample were obtained and stored at deepfreezer. All samples obtained at each point were tested for thequantification of EPO by EPO ELISA kit (R&D, cat #. DEP00). As shown inFIG. 8( a), all individual monkeys injected with EPO-hFc-1 or EPO-hFc-5via SC or IV routes showed longer half life than individual monkeysinjected with Aranesp® via SC or IV routes.

To investigate the pharmacokinetics of G-CSF-hFc-1, 100 ug/kg ofLEUCOSTIM® (filgrastim, DongA, Republic of Korea) as a control andG-CSF-hFc-1 were administrated via SC or IV routes to two male SpragueDawley Rats (Charles River Laboratories, Wilmington) per group. Bloodwas obtained before injection and 1, 2, 3, 4, 8, 12, 24, 48, 72, 96, 120and 192 h post-injection. Sera obtained by centrifugation at 3,000 rpmfor 10 min after incubation at room temperature for 30 min and stored atdeep freezer. Samples were quantified with several dilution folds suchas 1/2, 1/5, 1/50, 1/250, 1/500 using G-CSF kit (Biosource, Camarillo,#KHC2032). As shown in FIG. 8( b), G-CSF-hFc-1 injected via SC or IVroutes showed longer half-life than LEUCOSTIM®. G-CSF-hFc-1 and G-CSFhad 8.76 h and 2.36 h of in vivo t_(1/2) after SC administration and10.42 h and 1.78 h after IV administration, respectively. Therefore,G-CSF-hFc-1 showed an enhancement of 3.7-fold following SC injectionsand 5.9-fold following IV injection, compared to the LEUCOSTIM®.

To investigate the pharmacokinetics of p40N303Q-hFc-5 and Enbrel®, threecynomolgus monkeys per group were treated with a single SC injection ina dose of 100 ug/kg. Blood samples of each monkey were obtained beforeinjection and at 8, 24, 48, 72, 96, 120, 168, 336, 504, and 672 hpost-injection. Blood samples were incubated at room temperature for 30min to be clotted. After centrifugation at 3000 rpm for 10 min, serafrom each sample were obtained and stored at deep freezer. All samplesobtained at each point were tested for the quantification of human p40and human TNFR II by ELISA kits (R&D system, Minneapolis, #DY1240 and#DRT200, respectively). As shown in FIG. 8( c), p40N303Q-hFc-5 showedlonger half life than Enbrel® (average 199 h vs 127 h), althoughp40N303Q-hFc-5 showed lower Cmax value than Enbrel® (average 3 ng/ml vs7 ng/ml).

To investigate the pharmacokinetics of TNFR-hFc-5 and Enbrel®, threemale Sprague Dawley Rats (Charles River Laboratories, Wilmington) pergroup were treated with a single SC injection in a dose of 500 ug/kg.Blood samples of each rat were obtained before injection and at 2, 4, 8,12, 24, 30, 48, 72 and 120 h post-injection. Blood samples wereincubated at room temperature for 30 min to be clotted. Aftercentrifugation at 3,000 rpm for 10 min, sera from each sample wereobtained and stored at deep freezer. All samples obtained at each pointwere tested for the quantification of human TNFR II by ELISA kits (R&Dsystem, Minneapolis, #DRT200). As shown in FIG. 8( d), TNFR-hFc-5 showedslightly higher AUC level than Enbrel® (average 198.1 vs 172.9 ug*h/ml),although TNFR-hFc-5 showed similar half life to Enbrel® (average 28.6 hvs 29.4 h).

Example 8 In Vivo Bioactivity of Purified hFc-Fused Proteins

To compare the in vivo bioactivity of EPO-hFc-5 and Aranesp®, threecynomolgus monkeys per group were treated with a SC injection or asingle IV injection in a dose of 2,400 IU/kg. Blood samples of eachmonkey were obtained before injection and at 1, 3, 6, 12, 24, 30, 48,54, 72, 78, 96, 120, 168, 336, 504, and 672 h post-injection. The numberof various blood cells including reticulocytes was measured to evaluatethe in vivo bioactivity of EPO-hFc-5 and Aranesp®. As shown in FIG. 9(a), EPO-hFc-5 showed slightly higher in vitro potency than Aranesp® inboth SC and IV routes in terms of increase of reticulocytes in monkeys.

To investigate the in vivo bioactivity of G-CSF-hFc-1, LEUCOSTIM®(filgrastim, DongA, Republic of Korea) as a control and G-CSF-hFc-1 in adose of 100 ug/kg were administrated via SC or IV routes to two maleSprague Dawley Rats (Charles River Laboratories, Wilmington) per group.Blood was obtained using EDTA tube before injection and 1, 2, 3, 4, 8,12, 24, 48, 72, 96, 120 and 192 h post-injection. Each blood sample wastreated with RBC lysis buffer (BD Bioscience, Korea) for 4 minutes andtotal WBCs (white blood cells) diluted in FACS buffer were countedrepeatedly three times using hematocytometer. The number of granulocytewas measured using FACS caliber by determination of cell size by FSC(forward scatter) and granules by SSC (side scatter). As illustrated inFIG. 9( b), LEUCOSTIM® treated via SC and IV routes induced the peaknumber of WBC and granulocyte at 24 hours post-injection, whileG-CFS-hFc-1 induced the peak number of WBC and granulocytes at 72 hourspost-SC injection and 48 hours post-IV injection. From 24 h to 120 hafter injection, G-CSF-hFc-1 had more sustained in vivo bioactivity,compared with LEUCOSTIM®.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

What is claimed is:
 1. A chimeric polypeptide comprising a hybrid humanFc; and an granulocyte colony stimulating factor (“G-CSF”), a variant orfragment thereof, coupled to the hybrid human Fc, wherein said hybridhuman Fc is represented by the following formula:N′—(Z1)_(p)-Y—Z2-Z3-Z4-C′ wherein: N′ is the N-terminus and C′ is theC-terminus of the polypeptide, Y is an amino acid sequence consisting of5 or more consecutive amino acid residues starting from the C-terminalof the sequence of positions 99 to 162 of SEQ ID NO: 14; Z2 is an aminoacid sequence consisting of 4 or more consecutive amino acid residuesstarting from the N-terminal of the sequence of positions 163 to 199 ofSEQ ID NO: 14; Z3 is an amino acid sequence consisting of 71 or moreconsecutive amino acid residues starting from the C-terminal of thesequence of positions 115 to 220 of SEQ ID NO: 13; Z4 is an amino acidsequence consisting of 80 or more consecutive amino acid residuesstarting from the N-terminal of the sequence of positions 221 to 327 ofSEQ ID NO: 13; Z1 is an amino acid sequence consisting of 5 or moreconsecutive amino acid residues starting from the C-terminal of thesequence of positions 90 to 98 of (i) SEQ ID NO: 11 or (ii) SEQ ID NO:14; and p is an integer of 0 or 1, wherein the total number of the aminoacid residues for Z2 and Z3 is between 80 and 140, both inclusive;wherein the total number of the amino acid residues for the polypeptideis between 154 and 288, both inclusive; and wherein the G-CSF, itsvariant or fragment is fused at the N-terminus or the C-terminus of thehybrid human Fc, and wherein said G-CSF, its variant or fragment fusedto said hybrid human Fc shows an increased circulating half-lifecompared to the circulating half-life of said G-CSF, its variant orfragment without being fused to hybrid human Fc, and possesses G-CSFactivity.
 2. The chimeric polypeptide of claim 1, wherein Y is an aminoacid sequence consisting of the amino acid residues at positions 158 to162 of SEQ ID NO: 14, amino acid residues at positions 153 to 162 of SEQID NO: 14, amino acid residues at positions 143 to 162 of SEQ ID NO: 14,amino acid residues at positions 133 to 162 of SEQ ID NO: 14, or aminoacid residues at positions 99 to 162 of SEQ ID NO:
 14. 3. The chimericpolypeptide of claim 1, wherein Z2 is an amino acid sequence consistingof the amino acid residues at positions 163 to 170 of SEQ ID NO:
 14. 4.The chimeric polypeptide of any one of claims 1-3, wherein Z3 is anamino acid sequence consisting of the amino acid residues at positions121 to 220 of SEQ ID NO:
 13. 5. The chimeric polypeptide of claim 1,wherein Z4 is an amino acid sequence consisting of the amino acidresidues at positions 221 to 327 of SEQ ID NO:
 13. 6. The chimericpolypeptide of claim 4, wherein Z4 is an amino acid sequence consistingof the amino acid residues at positions 221 to 327 of SEQ ID NO:
 13. 7.The chimeric polypeptide of claim 1, wherein p is
 0. 8. The chimericpolypeptide of claim 1, wherein the hybrid human Fc and the G-CSF, itsvariant or fragment are coupled to each other via a linker selected fromthe group consisting of an albumin linker and a synthetic linker.
 9. Thechimeric polypeptide of claim 8, wherein said albumin linker comprisesamino acid sequence 321 to 323, 318 to 325, 316 to 328, 313 to 330, 311to 333, or 306 to 338 of SEQ ID NO:
 25. 10. The chimeric polypeptide ofclaim 8, wherein said synthetic linker is a peptide of 10 to 20 aminoacid residues, wherein the peptide being composed of Gly and Serresidues.
 11. The chimeric polypeptide of claim 1, wherein the hybridhuman Fc is encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 26 and SEQ ID NO:
 27. 12. Thechimeric polypeptide of claim 1, wherein the hybrid human Fc has anamino acid sequence selected from the group consisting of SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ IDNO: 23, SEQ ID NO: 28 and SEQ ID NO:
 29. 13. The chimeric polypeptide ofclaim 1, wherein the hybrid human Fc has the amino acid sequence of SEQID NO:
 22. 14. A nucleic acid molecule encoding the polypeptide ofclaim
 1. 15. An expression vector comprising the nucleic acid moleculeaccording to claim
 14. 16. A method of producing the chimericpolypeptide of claim 1, wherein the method comprises the steps of: (i)introducing a nucleic acid molecule encoding the chimeric polypeptide ofclaim 1 into a mammalian host cell, (ii) growing the cell in a mediumunder conditions where the polypeptide can be expressed; and (iii)harvesting the expressed chimeric polypeptide from the cell or themedium.
 17. A method for increasing a circulating half-life of angranulocyte colony stimulating factor (“G-CSF”), its variant or fragmentin a subject, comprising administering the chimeric polypeptide of claim1 to the subject.
 18. The method of claim 17, wherein the hybrid humanFc and the G-CSF, its variant or fragment are coupled to each other viaa linker selected from the group consisting of an albumin linker and asynthetic linker.
 19. A method for treating or ameliorating symptomsassociated with depletion of colony stimulating factor, the methodcomprising administering an effective amount of a composition comprisingthe chimeric polypeptide of claim 1 to a subject in need thereof. 20.The method of claim 19, wherein symptoms are chemotherapy-inducedneutropenia myelosuppression after bone marrow transplantation, acuteleukemia, aplastic anemia, myelodysplastic syndrome, or severe chronicneutropenias.